Compositions and methods for the therapy and diagnosis of lung cancer

ABSTRACT

Compositions and methods for the therapy and diagnosis of cancer, particularly lung cancer, are disclosed. Illustrative compositions comprise one or more lung tumor polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of diseases, particularly lung cancer.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 210121 455C26 SEQUENCE LISTING.txt. The textfile is 610 KB, was created on Jun. 7, 2010, and is being submittedelectronically via EFS-Web, concurrent with the filing of thespecification.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to therapy and diagnosis ofcancer, such as lung cancer. The invention is more specifically relatedto polypeptides, comprising at least a portion of a lung tumor protein,and to polynucleotides encoding such polypeptides. Such polypeptides andpolynucleotides are useful in pharmaceutical compositions, e.g.,vaccines, and other compositions for the diagnosis and treatment of lungcancer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Cancer is a significant health problem throughout the world. Althoughadvances have been made in detection and therapy of cancer, no vaccineor other universally successful method for prevention and/or treatmentis currently available. Current therapies, which are generally based ona combination of chemotherapy or surgery and radiation, continue toprove inadequate in many patients.

2. Description of Related Art

Lung cancer is the primary cause of cancer death among both men andwomen in the U.S., with an estimated 172,000 new cases being reported in1994. The five-year survival rate among all lung cancer patients,regardless of the stage of disease at diagnosis, is only 13%. Thiscontrasts with a five-year survival rate of 46% among cases detectedwhile the disease is still localized. However, only 16% of lung cancersare discovered before the disease has spread.

In spite of considerable research into therapies for these and othercancers, lung cancer remains difficult to diagnose and treateffectively. Accordingly, there is a need in the art for improvedmethods for detecting and treating such cancers. The present inventionfulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides polynucleotidecompositions comprising a sequence selected from the group consistingof:

(a) sequences provided in SEQ ID NO:1-3, 6-8, 10-13, 15-27, 29, 30, 32,34-49, 51, 52, 54, 55, 57-59, 61-69, 71, 73, 74, 77, 78, 80-82, 84,86-96, 107-109, 111, 113, 125, 127, 128, 129, 131-133, 142, 144,148-151, 153, 154, 157, 158, 160, 167, 168, 171, 179, 182, 184-186,188-191, 193, 194, 198-207, 209, 210, 213, 214, 217, 220-224, 253-337,345, 347, 349, 358, 362, 364, 365, 368, 370-375, 420, 424, 428, 431,434, 442, 447, 450, 467, 478, 479, 483, 485, and 489;

(b) complements of the sequences provided in SEQ ID NO:1-3, 6-8, 10-13,15-27, 29, 30, 32, 34-49, 51, 52, 54, 55, 57-59, 61-69, 71, 73, 74, 77,78, 80-82, 84, 86-96, 107-109, 111, 113, 125, 127, 128, 129, 131-133,142, 144, 148-151, 153, 154, 157, 158, 160, 167, 168, 171, 179, 182,184-186, 188-191, 193, 194, 198-207, 209, 210, 213, 214, 217, 220-224,253-337, 345, 347, 349, 358, 362, 364, 365, 368, 370-375, 420, 424, 428,431, 434, 442, 447, 450, 467, 478, 479, 483, 485, and 489;

(c) sequences consisting of at least 10, 15, 20, 25, 30, 35, 40, 45, 50,75 and 100 contiguous residues of a sequence provided in SEQ ID NO:1-3,6-8, 10-13, 15-27, 29, 30, 32, 34-49, 51, 52, 54, 55, 57-59, 61-69, 71,73, 74, 77, 78, 80-82, 84, 86-96, 107-109, 111, 113, 125, 127, 128, 129,131-133, 142, 144, 148-151, 153, 154, 157, 158, 160, 167, 168, 171, 179,182, 184-186, 188-191, 193, 194, 198-207, 209, 210, 213, 214, 217,220-224, 253-337, 345, 347, 349, 358, 362, 364, 365, 368, 370-375, 420,424, 428, 431, 434, 442, 447, 450, 467, 478, 479, 483, 485, and 489;

(d) sequences that hybridize to a sequence provided in SEQ ID NO:1-3,6-8, 10-13, 15-27, 29, 30, 32, 34-49, 51, 52, 54, 55, 57-59, 61-69, 71,73, 74, 77, 78, 80-82, 84, 86-96, 107-109, 111, 113, 125, 127, 128, 129,131-133, 142, 144, 148-151, 153, 154, 157, 158, 160, 167, 168, 171, 179,182, 184-186, 188-191, 193, 194, 198-207, 209, 210, 213, 214, 217,220-224, 253-337, 345, 347, 349, 358, 362, 364, 365, 368, 370-375, 420,424, 428, 431, 434, 442, 447, 450, 467, 478, 479, 483, 485, and 489,under moderate or highly stringent conditions;

(e) sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identity to a sequence of SEQ ID NO:1-3, 6-8, 10-13, 15-27, 29, 30,32, 34-49, 51, 52, 54, 55, 57-59, 61-69, 71, 73, 74, 77, 78, 80-82, 84,86-96, 107-109, 111, 113, 125, 127, 128, 129, 131-133, 142, 144,148-151, 153, 154, 157, 158, 160, 167, 168, 171, 179, 182, 184-186,188-191, 193, 194, 198-207, 209, 210, 213, 214, 217, 220-224, 253-337,345, 347, 349, 358, 362, 364, 365, 368, 370-375, 420, 424, 428, 431,434, 442, 447, 450, 467, 478, 479, 483, 485, and 489; and

(f) degenerate variants of a sequence provided in SEQ ID NO:1-3, 6-8,10-13, 15-27, 29, 30, 32, 34-49, 51, 52, 54, 55, 57-59, 61-69, 71, 73,74, 77, 78, 80-82, 84, 86-96, 107-109, 111, 113, 125, 127, 128, 129,131-133, 142, 144, 148-151, 153, 154, 157, 158, 160, 167, 168, 171, 179,182, 184-186, 188-191, 193, 194, 198-207, 209, 210, 213, 214, 217,220-224, 253-337, 345, 347, 349, 358, 362, 364, 365, 368, 370-375, 420,424, 428, 431, 434, 442, 447, 450, 467, 478, 479, 483, 485, and 489.

In one preferred embodiment, the polynucleotide compositions of theinvention are expressed in at least about 20%, more preferably in atleast about 30%, and most preferably in at least about 50% of lungtumors samples tested, at a level that is at least about 2-fold,preferably at least about 5-fold, and most preferably at least about10-fold higher than that for normal tissues.

The present invention, in another aspect, provides polypeptidecompositions comprising an amino acid sequence that is encoded by apolynucleotide sequence described above.

The present invention further provides polypeptide compositionscomprising an amino acid sequence selected from the group consisting ofsequences recited in SEQ ID NO:152, 155, 156, 165, 166, 169, 170, 172,174, 176, 226-252, 338-344, 346, 350, 357, 361, 363, 365, 367, 369,376-382, 387-419, 423, 427, 430, 433, 441, 443, 446, 449, 451-466,468-477, 480-482, 484, 486, 490-560 and 561-563.

In certain preferred embodiments, the polypeptides and/orpolynucleotides of the present invention are immunogenic, i.e., they arecapable of eliciting an immune response, particularly a humoral and/orcellular immune response, as further described herein.

The present invention further provides fragments, variants and/orderivatives of the disclosed polypeptide and/or polynucleotidesequences, wherein the fragments, variants and/or derivatives preferablyhave a level of immunogenic activity of at least about 50%, preferablyat least about 70% and more preferably at least about 90% of the levelof immunogenic activity of a polypeptide sequence set forth in SEQ IDNO:152, 155, 156, 165, 166, 169, 170, 172, 174, 176, 226-252, 338-344,346, 350, 357, 361, 363, 365, 367, 369, 376-382, 387-419, 423, 427, 430,433, 441, 443, 446, 449, 451-466, 486, 490-560 and 561-563, or apolypeptide sequence encoded by a polynucleotide sequence set forth inSEQ ID NO:1-3, 6-8, 10-13, 15-27, 29, 30, 32, 34-49, 51, 52, 54, 55,57-59, 61-69, 71, 73, 74, 77, 78, 80-82, 84, 86-96, 107-109, 111, 113,125, 127, 128, 129, 131-133, 142, 144, 148-151, 153, 154, 157, 158, 160,167, 168, 171, 179, 182, 184-186, 188-191, 193, 194, 198-207, 209, 210,213, 214, 217, 220-224, 253-337, 345, 347, 349, 358, 362, 364, 365, 368,370-375, 420, 424, 428, 431, 434, 442, 447, 450, 467, 478, 479, 483,485, and 489.

The present invention further provides polynucleotides that encode apolypeptide described above, expression vectors comprising suchpolynucleotides and host cells transformed or transfected with suchexpression vectors.

Within other aspects, the present invention provides pharmaceuticalcompositions comprising a polypeptide or polynucleotide as describedabove and a physiologically acceptable carrier.

Within a related aspect of the present invention, the pharmaceuticalcompositions, e.g., vaccine compositions, are provided for prophylacticor therapeutic applications. Such compositions generally comprise animmunogenic polypeptide or polynucleotide of the invention and animmunostimulant, such as an adjuvant.

The present invention further provides pharmaceutical compositions thatcomprise: (a) an antibody or antigen-binding fragment thereof thatspecifically binds to a polypeptide of the present invention, or afragment thereof; and (b) a physiologically acceptable carrier.

Within further aspects, the present invention provides pharmaceuticalcompositions comprising: (a) an antigen presenting cell that expresses apolypeptide as described above and (b) a pharmaceutically acceptablecarrier or excipient. Illustrative antigen presenting cells includedendritic cells, macrophages, monocytes, fibroblasts and B cells.

Within related aspects, pharmaceutical compositions are provided thatcomprise: (a) an antigen presenting cell that expresses a polypeptide asdescribed above and (b) an immunostimulant.

The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins, typically in theform of pharmaceutical compositions, e.g., vaccine compositions,comprising a physiologically acceptable carrier and/or animmunostimulant. The fusions proteins may comprise multiple immunogenicpolypeptides or portions/variants thereof, as described herein, and mayfurther comprise one or more polypeptide segments for facilitating theexpression, purification and/or immunogenicity of the polypeptide(s).

Within further aspects, the present invention provides methods forstimulating an immune response in a patient, preferably a T cellresponse in a human patient, comprising administering a pharmaceuticalcomposition described herein. The patient may be afflicted with lungcancer, in which case the methods provide treatment for the disease, orpatient considered at risk for such a disease may be treatedprophylactically.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient a pharmaceutical composition as recitedabove. The patient may be afflicted with lung cancer, in which case themethods provide treatment for the disease, or patient considered at riskfor such a disease may be treated prophylactically.

The present invention further provides, within other aspects, methodsfor removing tumor cells from a biological sample, comprising contactinga biological sample with T cells that specifically react with apolypeptide of the present invention, wherein the step of contacting isperformed under conditions and for a time sufficient to permit theremoval of cells expressing the protein from the sample.

Within related aspects, methods are provided for inhibiting thedevelopment of a cancer in a patient, comprising administering to apatient a biological sample treated as described above.

Methods are further provided, within other aspects, for stimulatingand/or expanding T cells specific for a polypeptide of the presentinvention, comprising contacting T cells with one or more of: (i) apolypeptide as described above; (ii) a polynucleotide encoding such apolypeptide; and/or (iii) an antigen presenting cell that expresses sucha polypeptide; under conditions and for a time sufficient to permit thestimulation and/or expansion of T cells. Isolated T cell populationscomprising T cells prepared as described above are also provided.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient an effective amount of a T cell population asdescribed above.

The present invention further provides methods for inhibiting thedevelopment of a cancer in a patient, comprising the steps of: (a)incubating CD4⁺ and/or CD8⁺ T cells isolated from a patient with one ormore of: (i) a polypeptide comprising at least an immunogenic portion ofpolypeptide disclosed herein; (ii) a polynucleotide encoding such apolypeptide; and (iii) an antigen-presenting cell that expressed such apolypeptide; and (b) administering to the patient an effective amount ofthe proliferated T cells, and thereby inhibiting the development of acancer in the patient. Proliferated cells may, but need not, be clonedprior to administration to the patient.

Within further aspects, the present invention provides methods fordetermining the presence or absence of a cancer, preferably a lungcancer, in a patient comprising: (a) contacting a biological sampleobtained from a patient with a binding agent that binds to a polypeptideas recited above; (b) detecting in the sample an amount of polypeptidethat binds to the binding agent; and (c) comparing the amount ofpolypeptide with a predetermined cut-off value, and therefromdetermining the presence or absence of a cancer in the patient. Withinpreferred embodiments, the binding agent is an antibody, more preferablya monoclonal antibody.

The present invention also provides, within other aspects, methods formonitoring the progression of a cancer in a patient. Such methodscomprise the steps of: (a) contacting a biological sample obtained froma patient at a first point in time with a binding agent that binds to apolypeptide as recited above; (b) detecting in the sample an amount ofpolypeptide that binds to the binding agent; (c) repeating steps (a) and(b) using a biological sample obtained from the patient at a subsequentpoint in time; and (d) comparing the amount of polypeptide detected instep (c) with the amount detected in step (b) and therefrom monitoringthe progression of the cancer in the patient.

The present invention further provides, within other aspects, methodsfor determining the presence or absence of a cancer in a patient,comprising the steps of: (a) contacting a biological sample obtainedfrom a patient with an oligonucleotide that hybridizes to apolynucleotide that encodes a polypeptide of the present invention; (b)detecting in the sample a level of a polynucleotide, preferably mRNA,that hybridizes to the oligonucleotide; and (c) comparing the level ofpolynucleotide that hybridizes to the oligonucleotide with apredetermined cut-off value, and therefrom determining the presence orabsence of a cancer in the patient. Within certain embodiments, theamount of mRNA is detected via polymerase chain reaction using, forexample, at least one oligonucleotide primer that hybridizes to apolynucleotide encoding a polypeptide as recited above, or a complementof such a polynucleotide. Within other embodiments, the amount of mRNAis detected using a hybridization technique, employing anoligonucleotide probe that hybridizes to a polynucleotide that encodes apolypeptide as recited above, or a complement of such a polynucleotide.

In related aspects, methods are provided for monitoring the progressionof a cancer in a patient, comprising the steps of: (a) contacting abiological sample obtained from a patient with an oligonucleotide thathybridizes to a polynucleotide that encodes a polypeptide of the presentinvention; (b) detecting in the sample an amount of a polynucleotidethat hybridizes to the oligonucleotide; (c) repeating steps (a) and (b)using a biological sample obtained from the patient at a subsequentpoint in time; and (d) comparing the amount of polynucleotide detectedin step (c) with the amount detected in step (b) and therefrommonitoring the progression of the cancer in the patient.

Within further aspects, the present invention provides antibodies, suchas monoclonal antibodies, that bind to a polypeptide as described above,as well as diagnostic kits comprising such antibodies. Diagnostic kitscomprising one or more oligonucleotide probes or primers as describedabove are also provided.

A further aspect of the present invention provides methods for detectingthe presence or absence of a cervical cancer in a patient, comprisingcontacting a test cervical tissue sample obtained from the patient withan antibody that specifically binds to the polypeptide set forth in SEQID NO:176; detecting an amount of the antibody that binds to thepolypeptide in the cervical tissue sample;

and comparing the amount of the antibody that binds to the polypeptidein the test cervical tissue sample to the amount of the antibody thatbinds to the polypeptide in a control cervical tissue sample; andthereby detecting the presence or absence of a cervical cancer in thepatient. In certain embodiments, the amount of antibody that binds tothe polypeptide in the test cervical tissue sample and the controlcervical tissue sample is determined using immunohistochemistry. In thisregard, antibody staining in at least 25% of cells indicates thepresence of a cervical cancer in the patient. In another embodiment, theamount of antibody that binds to the polypeptide in the test cervicaltissue sample and the control cervical tissue is determined using anELISA. In this regard, at least a two-fold increase in the amount of theantibody that binds to the polypeptide in the cervical tissue sample ascompared to the amount of the antibody that binds to the polypeptide inthe control cervical tissue sample indicates the presence of a cervicalcancer in the patient. In a further embodiment, the antibody is amonoclonal antibody, such as a murine monoclonal antibody. Otherappropriate antibodies that are specific for the L523S polypeptide asset forth in SEQ ID NO:176 are contemplated herein. In yet a furtherembodiment, the test cervical tissue sample is obtained from a patientsuspected of having AIS of the uterine cervix or ECCA. In an additionalembodiment, the control cervical tissue sample comprises a benignendocervical gland sample or an endometrial adenocarcinoma sample. Otherappropriate test and control samples may also be used.

A further aspect of the present invention provides a method fordetermining whether a patient is at risk for developing a cervicalcancer, comprising contacting a test cervical tissue sample obtainedfrom the patient with an antibody that specifically binds to thepolypeptide set forth in SEQ ID NO:176; detecting an amount of theantibody that binds to the polypeptide in the cervical tissue sample;and comparing the amount of the antibody that binds to the polypeptidein the test cervical tissue sample to the amount of the antibody thatbinds to the polypeptide in a control cervical tissue sample; andthereby determining whether a patient is at risk for developing acervical cancer. In one embodiment, the test cervical tissue samplecomprises a cervical intraepithelial neoplasia.

These and other aspects of the present invention will become apparentupon reference to the following detailed description. All referencesdisclosed herein are hereby incorporated by reference in their entiretyas if each was incorporated individually.

A BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

SEQ ID NO:1 is the determined cDNA sequence for LST-S1-2

SEQ ID NO:2 is the determined cDNA sequence for LST-S1-28

SEQ ID NO:3 is the determined cDNA sequence for LST-S1-90

SEQ ID NO:4 is the determined cDNA sequence for LST-S1-144

SEQ ID NO:5 is the determined cDNA sequence for LST-S1-133

SEQ ID NO:6 is the determined cDNA sequence for LST-S1-169

SEQ ID NO:7 is the determined cDNA sequence for LST-S2-6

SEQ ID NO:8 is the determined cDNA sequence for LST-S2-11

SEQ ID NO:9 is the determined cDNA sequence for LST-S2-17

SEQ ID NO:10 is the determined cDNA sequence for LST-S2-25

SEQ ID NO:11 is the determined cDNA sequence for LST-S2-39

SEQ ID NO:12 is a first determined cDNA sequence for LST-S2-43

SEQ ID NO:13 is a second determined cDNA sequence for LST-S2-43

SEQ ID NO:14 is the determined cDNA sequence for LST-S2-65

SEQ ID NO:15 is the determined cDNA sequence for LST-S2-68

SEQ ID NO:16 is the determined cDNA sequence for LST-S2-72

SEQ ID NO:17 is the determined cDNA sequence for LST-S2-74

SEQ ID NO:18 is the determined cDNA sequence for LST-S2-103

SEQ ID NO:19 is the determined cDNA sequence for LST-S2-N1-1F

SEQ ID NO:20 is the determined cDNA sequence for LST-S2-N1-2A

SEQ ID NO:21 is the determined cDNA sequence for LST-S2-N1-4H

SEQ ID NO:22 is the determined cDNA sequence for LST-S2-N1-5A

SEQ ID NO:23 is the determined cDNA sequence for LST-S2-N1-6B

SEQ ID NO:24 is the determined cDNA sequence for LST-S2-N1-7B

SEQ ID NO:25 is the determined cDNA sequence for LST-S2-N1-7H

SEQ ID NO:26 is the determined cDNA sequence for LST-S2-N1-8A

SEQ ID NO:27 is the determined cDNA sequence for LST-S2-N1-8D

SEQ ID NO:28 is the determined cDNA sequence for LST-S2-N1-9A

SEQ ID NO:29 is the determined cDNA sequence for LST-S2-N1-9E

SEQ ID NO:30 is the determined cDNA sequence for LST-S2-N1-10A

SEQ ID NO:31 is the determined cDNA sequence for LST-S2-N1-10G

SEQ ID NO:32 is the determined cDNA sequence for LST-S2-N1-11A

SEQ ID NO:33 is the determined cDNA sequence for LST-S2-N1-12C

SEQ ID NO:34 is the determined cDNA sequence for LST-S2-N1-12E

SEQ ID NO:35 is the determined cDNA sequence for LST-S2-B1-3D

SEQ ID NO:36 is the determined cDNA sequence for LST-S2-B1-6C

SEQ ID NO:37 is the determined cDNA sequence for LST-S2-B1-5D

SEQ ID NO:38 is the determined cDNA sequence for LST-S2-B1-5F

SEQ ID NO:39 is the determined cDNA sequence for LST-S2-B1-6G

SEQ ID NO:40 is the determined cDNA sequence for LST-S2-B1-8A

SEQ ID NO:41 is the determined cDNA sequence for LST-S2-B1-8D

SEQ ID NO:42 is the determined cDNA sequence for LST-S2-B1-10A

SEQ ID NO:43 is the determined cDNA sequence for LST-S2-B1-9B

SEQ ID NO:44 is the determined cDNA sequence for LST-S2-B1-9F

SEQ ID NO:45 is the determined cDNA sequence for LST-S2-B1-12D

SEQ ID NO:46 is the determined cDNA sequence for LST-S2-I2-2B

SEQ ID NO:47 is the determined cDNA sequence for LST-S2-I2-5F

SEQ ID NO:48 is the determined cDNA sequence for LST-S2-I2-6B

SEQ ID NO:49 is the determined cDNA sequence for LST-S2-I2-7F

SEQ ID NO:50 is the determined cDNA sequence for LST-S2-I2-8G

SEQ ID NO:51 is the determined cDNA sequence for LST-S2-I2-9E

SEQ ID NO:52 is the determined cDNA sequence for LST-S2-I2-12B

SEQ ID NO:53 is the determined cDNA sequence for LST-S2-H2-2C

SEQ ID NO:54 is the determined cDNA sequence for LST-S2-H2-1G

SEQ ID NO:55 is the determined cDNA sequence for LST-S2-H2-4G

SEQ ID NO:56 is the determined cDNA sequence for LST-S2-H2-3H

SEQ ID NO:57 is the determined cDNA sequence for LST-S2-H2-5G

SEQ ID NO:58 is the determined cDNA sequence for LST-S2-H2-9B

SEQ ID NO:59 is the determined cDNA sequence for LST-S2-H2-10H

SEQ ID NO:60 is the determined cDNA sequence for LST-S2-H2-12D

SEQ ID NO: 61 is the determined cDNA sequence for LST-S3-2

SEQ ID NO: 62 is the determined cDNA sequence for LST-S3-4

SEQ ID NO: 63 is the determined cDNA sequence for LST-S3-7

SEQ ID NO: 64 is the determined cDNA sequence for LST-S3-8

SEQ ID NO: 65 is the determined cDNA sequence for LST-S3-12

SEQ ID NO: 66 is the determined cDNA sequence for LST-S3-13

SEQ ID NO: 67 is the determined cDNA sequence for LST-S3-14

SEQ ID NO: 68 is the determined cDNA sequence for LST-S3-16

SEQ ID NO: 69 is the determined cDNA sequence for LST-S3-21

SEQ ID NO: 70 is the determined cDNA sequence for LST-S3-22

SEQ ID NO: 71 is the determined cDNA sequence for LST-S1-7

SEQ ID NO: 72 is the determined cDNA sequence for LST-S1-A-1E

SEQ ID NO: 73 is the determined cDNA sequence for LST-S1-A-1G

SEQ ID NO: 74 is the determined cDNA sequence for LST-S1-A-3E

SEQ ID NO: 75 is the determined cDNA sequence for LST-S1-A-4E

SEQ ID NO: 76 is the determined cDNA sequence for LST-S1-A-6D

SEQ ID NO: 77 is the determined cDNA sequence for LST-S1-A-8D

SEQ ID NO: 78 is the determined cDNA sequence for LST-S1-A-10A

SEQ ID NO: 79 is the determined cDNA sequence for LST-S1-A-100

SEQ ID NO: 80 is the determined cDNA sequence for LST-S1-A-9D

SEQ ID NO: 81 is the determined cDNA sequence for LST-S1-A-10D

SEQ ID NO: 82 is the determined cDNA sequence for LST-S1-A-9H

SEQ ID NO: 83 is the determined cDNA sequence for LST-S1-A-11D

SEQ ID NO: 84 is the determined cDNA sequence for LST-S1-A-12D

SEQ ID NO: 85 is the determined cDNA sequence for LST-S1-A-11E

SEQ ID NO: 86 is the determined cDNA sequence for LST-S1-A-12E

SEQ ID NO: 87 is the determined cDNA sequence for L513S (T3).

SEQ ID NO: 88 is the determined cDNA sequence for L513S contig 1.

SEQ ID NO: 89 is a first determined cDNA sequence for L514S.

SEQ ID NO: 90 is a second determined cDNA sequence for L514S.

SEQ ID NO: 91 is a first determined cDNA sequence for L516S.

SEQ ID NO: 92 is a second determined cDNA sequence for L516S.

SEQ ID NO: 93 is the determined cDNA sequence for L517S.

SEQ ID NO: 94 is the extended cDNA sequence for LST-S1-169 (also knownas L519S).

SEQ ID NO: 95 is a first determined cDNA sequence for L520S.

SEQ ID NO: 96 is a second determined cDNA sequence for L520S.

SEQ ID NO: 97 is a first determined cDNA sequence for L521S.

SEQ ID NO: 98 is a second determined cDNA sequence for L521S.

SEQ ID NO: 99 is the determined cDNA sequence for L522S.

SEQ ID NO: 100 is the determined cDNA sequence for L523S.

SEQ ID NO: 101 is the determined cDNA sequence for L524S.

SEQ ID NO: 102 is the determined cDNA sequence for L525S.

SEQ ID NO: 103 is the determined cDNA sequence for L526S.

SEQ ID NO: 104 is the determined cDNA sequence for L527S.

SEQ ID NO: 105 is the determined cDNA sequence for L528S.

SEQ ID NO: 106 is the determined cDNA sequence for L529S.

SEQ ID NO: 107 is a first determined cDNA sequence for L530S.

SEQ ID NO: 108 is a second determined cDNA sequence for L530S.

SEQ ID NO: 109 is the determined full-length cDNA sequence for L531Sshort form

SEQ ID NO: 110 is the amino acid sequence encoded by SEQ ID NO: 109.

SEQ ID NO: 111 is the determined full-length cDNA sequence for L531Slong form

SEQ ID NO: 112 is the amino acid sequence encoded by SEQ ID NO: 111.

SEQ ID NO: 113 is the determined full-length cDNA sequence for L520S.

SEQ ID NO: 114 is the amino acid sequence encoded by SEQ ID NO: 113.

SEQ ID NO: 115 is the determined cDNA sequence for contig 1.

SEQ ID NO: 116 is the determined cDNA sequence for contig 3.

SEQ ID NO: 117 is the determined cDNA sequence for contig 4.

SEQ ID NO: 118 is the determined cDNA sequence for contig 5.

SEQ ID NO: 119 is the determined cDNA sequence for contig 7.

SEQ ID NO: 120 is the determined cDNA sequence for contig 8.

SEQ ID NO: 121 is the determined cDNA sequence for contig 9.

SEQ ID NO: 122 is the determined cDNA sequence for contig 10.

SEQ ID NO: 123 is the determined cDNA sequence for contig 12.

SEQ ID NO: 124 is the determined cDNA sequence for contig 11.

SEQ ID NO: 125 is the determined cDNA sequence for contig 13 (also knownas L761P).

SEQ ID NO: 126 is the determined cDNA sequence for contig 15.

SEQ ID NO: 127 is the determined cDNA sequence for contig 16.

SEQ ID NO: 128 is the determined cDNA sequence for contig 17.

SEQ ID NO: 129 is the determined cDNA sequence for contig 19.

SEQ ID NO: 130 is the determined cDNA sequence for contig 20.

SEQ ID NO: 131 is the determined cDNA sequence for contig 22.

SEQ ID NO: 132 is the determined cDNA sequence for contig 24.

SEQ ID NO: 133 is the determined cDNA sequence for contig 29.

SEQ ID NO: 134 is the determined cDNA sequence for contig 31.

SEQ ID NO: 135 is the determined cDNA sequence for contig 33.

SEQ ID NO: 136 is the determined cDNA sequence for contig 38.

SEQ ID NO: 137 is the determined cDNA sequence for contig 39.

SEQ ID NO: 138 is the determined cDNA sequence for contig 41.

SEQ ID NO: 139 is the determined cDNA sequence for contig 43.

SEQ ID NO: 140 is the determined cDNA sequence for contig 44.

SEQ ID NO: 141 is the determined cDNA sequence for contig 45.

SEQ ID NO: 142 is the determined cDNA sequence for contig 47.

SEQ ID NO: 143 is the determined cDNA sequence for contig 48.

SEQ ID NO: 144 is the determined cDNA sequence for contig 49.

SEQ ID NO: 145 is the determined cDNA sequence for contig 50.

SEQ ID NO: 146 is the determined cDNA sequence for contig 53.

SEQ ID NO: 147 is the determined cDNA sequence for contig 54.

SEQ ID NO: 148 is the determined cDNA sequence for contig 56.

SEQ ID NO: 149 is the determined cDNA sequence for contig 57.

SEQ ID NO: 150 is the determined cDNA sequence for contig 58.

SEQ ID NO: 151 is the full-length cDNA sequence for L530S.

SEQ ID NO: 152 is the amino acid sequence encoded by SEQ ID NO: 151

SEQ ID NO: 153 is the full-length cDNA sequence of a first variant ofL514S

SEQ ID NO: 154 is the full-length cDNA sequence of a second variant ofL514S

SEQ ID NO: 155 is the amino acid sequence encoded by SEQ ID NO: 153.

SEQ ID NO: 156 is the amino acid sequence encoded by SEQ ID NO: 154.

SEQ ID NO: 157 is the determined cDNA sequence for contig 59.

SEQ ID NO: 158 is the full-length cDNA sequence for L763P (also referredto as contig 22).

SEQ ID NO: 159 is the amino acid sequence encoded by SEQ ID NO: 158.

SEQ ID NO: 160 is the full-length cDNA sequence for L762P (also referredto as contig 17).

SEQ ID NO: 161 is the amino acid sequence encoded by SEQ ID NO: 160.

SEQ ID NO: 162 is the determined cDNA sequence for L515S.

SEQ ID NO: 163 is the full-length cDNA sequence of a first variant ofL524S.

SEQ ID NO: 164 is the full-length cDNA sequence of a second variant ofL524S.

SEQ ID NO: 165 is the amino acid sequence encoded by SEQ ID NO: 163.

SEQ ID NO: 166 is the amino acid sequence encoded by SEQ ID NO: 164.

SEQ ID NO: 167 is the full-length cDNA sequence of a first variant ofL762P.

SEQ ID NO: 168 is the full-length cDNA sequence of a second variant ofL762P.

SEQ ID NO: 169 is the amino acid sequence encoded by SEQ ID NO: 167.

SEQ ID NO: 170 is the amino acid sequence encoded by SEQ ID NO: 168.

SEQ ID NO: 171 is the full-length cDNA sequence for L773P (also referredto as contig 56).

SEQ ID NO: 172 is the amino acid sequence encoded by SEQ ID NO: 171.

SEQ ID NO: 173 is an extended cDNA sequence for L519S.

SEQ ID NO: 174 is the amino acid sequence encoded by SEQ ID NO: 174.

SEQ ID NO: 175 is the full-length cDNA sequence for L523S.

SEQ ID NO: 176 is the amino acid sequence encoded by SEQ ID NO: 175.

SEQ ID NO: 177 is the determined cDNA sequence for LST-sub5-7A.

SEQ ID NO: 178 is the determined cDNA sequence for LST-sub5-8G.

SEQ ID NO: 179 is the determined cDNA sequence for LST-sub5-8H.

SEQ ID NO: 180 is the determined cDNA sequence for LST-sub5-10B.

SEQ ID NO: 181 is the determined cDNA sequence for LST-sub5-10H.

SEQ ID NO: 182 is the determined cDNA sequence for LST-sub5-12B.

SEQ ID NO: 183 is the determined cDNA sequence for LST-sub5-11C.

SEQ ID NO: 184 is the determined cDNA sequence for LST-sub6-1c.

SEQ ID NO: 185 is the determined cDNA sequence for LST-sub6-2f.

SEQ ID NO: 186 is the determined cDNA sequence for LST-sub6-2G.

SEQ ID NO: 187 is the determined cDNA sequence for LST-sub6-4d.

SEQ ID NO: 188 is the determined cDNA sequence for LST-sub6-4e.

SEQ ID NO: 189 is the determined cDNA sequence for LST-sub6-4f.

SEQ ID NO: 190 is the determined cDNA sequence for LST-sub6-3h.

SEQ ID NO: 191 is the determined cDNA sequence for LST-sub6-5d.

SEQ ID NO: 192 is the determined cDNA sequence for LST-sub6-5h.

SEQ ID NO: 193 is the determined cDNA sequence for LST-sub6-6h.

SEQ ID NO: 194 is the determined cDNA sequence for LST-sub6-7a.

SEQ ID NO: 195 is the determined cDNA sequence for LST-sub6-8a.

SEQ ID NO: 196 is the determined cDNA sequence for LST-sub6-7d.

SEQ ID NO: 197 is the determined cDNA sequence for LST-sub6-7e.

SEQ ID NO: 198 is the determined cDNA sequence for LST-sub6-8e.

SEQ ID NO: 199 is the determined cDNA sequence for LST-sub6-7g.

SEQ ID NO: 200 is the determined cDNA sequence for LST-sub6-9f.

SEQ ID NO: 201 is the determined cDNA sequence for LST-sub6-9h.

SEQ ID NO: 202 is the determined cDNA sequence for LST-sub6-11b.

SEQ ID NO: 203 is the determined cDNA sequence for LST-sub6-11c.

SEQ ID NO: 204 is the determined cDNA sequence for LST-sub6-12c.

SEQ ID NO: 205 is the determined cDNA sequence for LST-sub6-12e.

SEQ ID NO: 206 is the determined cDNA sequence for LST-sub6-12f.

SEQ ID NO: 207 is the determined cDNA sequence for LST-sub6-11g.

SEQ ID NO: 208 is the determined cDNA sequence for LST-sub6-12g.

SEQ ID NO: 209 is the determined cDNA sequence for LST-sub6-12h.

SEQ ID NO: 210 is the determined cDNA sequence for LST-sub6-II-1a.

SEQ ID NO: 211 is the determined cDNA sequence for LST-sub6-II-2b.

SEQ ID NO: 212 is the determined cDNA sequence for LST-sub6-II-2g.

SEQ ID NO: 213 is the determined cDNA sequence for LST-sub6-II-1h.

SEQ ID NO: 214 is the determined cDNA sequence for LST-sub6-II-4a.

SEQ ID NO: 215 is the determined cDNA sequence for LST-sub6-II-4b.

SEQ ID NO: 216 is the determined cDNA sequence for LST-sub6-II-3e.

SEQ ID NO: 217 is the determined cDNA sequence for LST-sub6-II-4f.

SEQ ID NO: 218 is the determined cDNA sequence for LST-sub6-II-4g.

SEQ ID NO: 219 is the determined cDNA sequence for LST-sub6-II-4h.

SEQ ID NO: 220 is the determined cDNA sequence for LST-sub6-II-5c.

SEQ ID NO: 221 is the determined cDNA sequence for LST-sub6-II-5e.

SEQ ID NO: 222 is the determined cDNA sequence for LST-sub6-II-6f.

SEQ ID NO: 223 is the determined cDNA sequence for LST-sub6-II-5g.

SEQ ID NO: 224 is the determined cDNA sequence for LST-sub6-II-6g.

SEQ ID NO: 225 is the amino acid sequence for L528S.

SEQ ID NO: 226-251 are synthetic peptides derived from L762P.

SEQ ID NO: 252 is the expressed amino acid sequence of L514S.

SEQ ID NO: 253 is the DNA sequence corresponding to SEQ ID NO: 252.

SEQ ID NO: 254 is the DNA sequence of a L762P expression construct.

SEQ ID NO: 255 is the determined cDNA sequence for clone 23785.

SEQ ID NO: 256 is the determined cDNA sequence for clone 23786.

SEQ ID NO: 257 is the determined cDNA sequence for clone 23788.

SEQ ID NO: 258 is the determined cDNA sequence for clone 23790.

SEQ ID NO: 259 is the determined cDNA sequence for clone 23793.

SEQ ID NO: 260 is the determined cDNA sequence for clone 23794.

SEQ ID NO: 261 is the determined cDNA sequence for clone 23795.

SEQ ID NO: 262 is the determined cDNA sequence for clone 23796.

SEQ ID NO: 263 is the determined cDNA sequence for clone 23797.

SEQ ID NO: 264 is the determined cDNA sequence for clone 23798.

SEQ ID NO: 265 is the determined cDNA sequence for clone 23799.

SEQ ID NO: 266 is the determined cDNA sequence for clone 23800.

SEQ ID NO: 267 is the determined cDNA sequence for clone 23802.

SEQ ID NO: 268 is the determined cDNA sequence for clone 23803.

SEQ ID NO: 269 is the determined cDNA sequence for clone 23804.

SEQ ID NO: 270 is the determined cDNA sequence for clone 23805.

SEQ ID NO: 271 is the determined cDNA sequence for clone 23806.

SEQ ID NO: 272 is the determined cDNA sequence for clone 23807.

SEQ ID NO: 273 is the determined cDNA sequence for clone 23808.

SEQ ID NO: 274 is the determined cDNA sequence for clone 23809.

SEQ ID NO: 275 is the determined cDNA sequence for clone 23810.

SEQ ID NO: 276 is the determined cDNA sequence for clone 23811.

SEQ ID NO: 277 is the determined cDNA sequence for clone 23812.

SEQ ID NO: 278 is the determined cDNA sequence for clone 23813.

SEQ ID NO: 279 is the determined cDNA sequence for clone 23815.

SEQ ID NO: 280 is the determined cDNA sequence for clone 25298.

SEQ ID NO: 281 is the determined cDNA sequence for clone 25299.

SEQ ID NO: 282 is the determined cDNA sequence for clone 25300.

SEQ ID NO: 283 is the determined cDNA sequence for clone 25301

SEQ ID NO: 284 is the determined cDNA sequence for clone 25304

SEQ ID NO: 285 is the determined cDNA sequence for clone 25309.

SEQ ID NO: 286 is the determined cDNA sequence for clone 25312.

SEQ ID NO: 287 is the determined cDNA sequence for clone 25317.

SEQ ID NO:288 is the determined cDNA sequence for clone 25321.

SEQ ID NO:289 is the determined cDNA sequence for clone 25323.

SEQ ID NO:290 is the determined cDNA sequence for clone 25327.

SEQ ID NO:291 is the determined cDNA sequence for clone 25328.

SEQ ID NO:292 is the determined cDNA sequence for clone 25332.

SEQ ID NO:293 is the determined cDNA sequence for clone 25333.

SEQ ID NO:294 is the determined cDNA sequence for clone 25336.

SEQ ID NO:295 is the determined cDNA sequence for clone 25340.

SEQ ID NO:296 is the determined cDNA sequence for clone 25342.

SEQ ID NO:297 is the determined cDNA sequence for clone 25356.

SEQ ID NO:298 is the determined cDNA sequence for clone 25357.

SEQ ID NO:299 is the determined cDNA sequence for clone 25361.

SEQ ID NO:300 is the determined cDNA sequence for clone 25363.

SEQ ID NO:301 is the determined cDNA sequence for clone 25397.

SEQ ID NO:302 is the determined cDNA sequence for clone 25402.

SEQ ID NO:303 is the determined cDNA sequence for clone 25403.

SEQ ID NO:304 is the determined cDNA sequence for clone 25405.

SEQ ID NO:305 is the determined cDNA sequence for clone 25407.

SEQ ID NO:306 is the determined cDNA sequence for clone 25409.

SEQ ID NO:307 is the determined cDNA sequence for clone 25396.

SEQ ID NO:308 is the determined cDNA sequence for clone 25414.

SEQ ID NO:309 is the determined cDNA sequence for clone 25410.

SEQ ID NO:310 is the determined cDNA sequence for clone 25406.

SEQ ID NO:311 is the determined cDNA sequence for clone 25306.

SEQ ID NO:312 is the determined cDNA sequence for clone 25362.

SEQ ID NO:313 is the determined cDNA sequence for clone 25360.

SEQ ID NO:314 is the determined cDNA sequence for clone 25398.

SEQ ID NO:315 is the determined cDNA sequence for clone 25355.

SEQ ID NO:316 is the determined cDNA sequence for clone 25351.

SEQ ID NO:317 is the determined cDNA sequence for clone 25331.

SEQ ID NO:318 is the determined cDNA sequence for clone 25338.

SEQ ID NO:319 is the determined cDNA sequence for clone 25335.

SEQ ID NO:320 is the determined cDNA sequence for clone 25329.

SEQ ID NO:321 is the determined cDNA sequence for clone 25324.

SEQ ID NO:322 is the determined cDNA sequence for clone 25322.

SEQ ID NO:323 is the determined cDNA sequence for clone 25319.

SEQ ID NO:324 is the determined cDNA sequence for clone 25316.

SEQ ID NO:325 is the determined cDNA sequence for clone 25311.

SEQ ID NO:326 is the determined cDNA sequence for clone 25310.

SEQ ID NO:327 is the determined cDNA sequence for clone 25302.

SEQ ID NO:328 is the determined cDNA sequence for clone 25315.

SEQ ID NO:329 is the determined cDNA sequence for clone 25308.

SEQ ID NO:330 is the determined cDNA sequence for clone 25303.

SEQ ID NO:331-337 are the cDNA sequences of isoforms of the p53 tumorsuppressor homologue, p63 (also referred to as L530S).

SEQ ID NO:338-344 are the amino acid sequences encoded by SEQ IDNO:331-337, respectively

SEQ ID NO:345 is a second cDNA sequence for the antigen L763P.

SEQ ID NO:346 is the amino acid sequence encoded by the sequence of SEQID NO: 345.

SEQ ID NO:347 is a determined full-length cDNA sequence for L523S.

SEQ ID NO:348 is the amino acid sequence encoded by SEQ ID NO: 347.

SEQ ID NO:349 is the cDNA sequence encoding the N-terminal portion ofL773P.

SEQ ID NO:350 is the amino acid sequence of the N-terminal portion ofL773P.

SEQ ID NO:351 is the DNA sequence for a fusion of Ra12 and theN-terminal portion of L763P.

SEQ ID NO:352 is the amino acid sequence of the fusion of Ra12 and theN-terminal portion of L763P.

SEQ ID NO:353 is the DNA sequence for a fusion of Ra12 and theC-terminal portion of L763P.

SEQ ID NO:354 is the amino acid sequence of the fusion of Ra12 and theC-terminal portion of L763P.

SEQ ID NO:355 is a primer.

SEQ ID NO:356 is a primer.

SEQ ID NO:357 is the protein sequence of expressed recombinant L762P.

SEQ ID NO:358 is the DNA sequence of expressed recombinant L762P.

SEQ ID NO:359 is a primer.

SEQ ID NO:360 is a primer.

SEQ ID NO:361 is the protein sequence of expressed recombinant L773P A.

SEQ ID NO:362 is the DNA sequence of expressed recombinant L773P A.

SEQ ID NO:363 is an epitope derived from clone L773P polypeptide.

SEQ ID NO:364 is a polynucleotide encoding the polypeptide of SEQ IDNO:363.

SEQ ID NO:365 is an epitope derived from clone L773P polypeptide.

SEQ ID NO:366 is a polynucleotide encoding the polypeptide of SEQ IDNO:365.

SEQ ID NO:367 is an epitope consisting of amino acids 571-590 of SEQ IDNO:161, clone L762P.

SEQ ID NO:368 is the full-length DNA sequence for contig 13 (SEQ IDNO:125), also referred to as L761P.

SEQ ID NO:369 is the protein sequence encoded by the DNA sequence of SEQID NO:368.

SEQ ID NO:370 is an L762P DNA sequence from nucleotides 2071-2130.

SEQ ID NO:371 is an L762P DNA sequence from nucleotides 1441-1500.

SEQ ID NO:372 is an L762P DNA sequence from nucleotides 1936-1955.

SEQ ID NO:373 is an L762P DNA sequence from nucleotides 2620-2679.

SEQ ID NO:374 is an L762P DNA sequence from nucleotides 1801-1860.

SEQ ID NO:375 is an L762P DNA sequence from nucleotides 1531-1591.

SEQ ID NO:376 is the amino acid sequence of the L762P peptide encoded bySEQ ID NO:373.

SEQ ID NO:377 is the amino acid sequence of the L762P peptide encoded bySEQ ID NO:370.

SEQ ID NO:378 is the amino acid sequence of the L762P peptide encoded bySEQ ID NO:372.

SEQ ID NO:379 is the amino acid sequence of the L762P peptide encoded bySEQ ID NO:374.

SEQ ID NO:380 is the amino acid sequence of the L762P peptide encoded bySEQ ID NO:371.

SEQ ID NO:381 is the amino acid sequence of the L762P peptide encoded bySEQ ID NO:375.

SEQ ID NO:382 is the amino acid sequence of an epitope of L762P.

SEQ ID NO:383-386 are PCR primers.

SEQ ID NO:387-395 are the amino acid sequences of L773P peptides.

SEQ ID NO:396-419 are the amino acid sequences of L5235 peptides.

SEQ ID NO:420 is the determined cDNA sequence for clone #19014.

SEQ ID NO:421 is the forward primer PDM-278 for the L514S-13160 codingregion.

SEQ ID NO:422 is the reverse primer PDM-278 for the L514S-13160 codingregion.

SEQ ID NO:423 is the amino acid sequence for the expressed recombinantL514S.

SEQ ID NO:424 is the DNA coding sequence for the recombinant L514S.

SEQ ID NO:425 is the forward primer PDM-414 for the L523S coding region.

SEQ ID NO:426 is the reverse primer PDM-414 for the L523S coding region.

SEQ ID NO:427 is the amino acid sequence for the expressed recombinantL523S.

SEQ ID NO:428 is the DNA coding sequence for the recombinant L523S.

SEQ ID NO:429 is the reverse primer PDM-279 for the L762PA codingregion.

SEQ ID NO:430 is the amino acid sequence for the expressed recombinantL762PA.

SEQ ID NO:431 is the DNA coding sequence for the recombinant L762PA.

SEQ ID NO:432 is the reverse primer PDM-300 for the L773P coding region.

SEQ ID NO:433 is the amino acid sequence of the expressed recombinantL773P.

SEQ ID NO:434 is the DNA coding sequence for the recombinant L773P.

SEQ ID NO:435 is the forward primer for TCR Valpha8.

SEQ ID NO:436 is the reverse primer for TCR Valpha8.

SEQ ID NO:437 is the forward primer for TCR Vbeta8.

SEQ ID NO:438 is the reverse primer for TCR Vbeta8.

SEQ ID NO:439 is the TCR Valpha DNA sequence of the TCR clone specificfor the lung antigen L762P.

SEQ ID NO:440 is the TCR Vbeta DNA sequence of the TCR clone specificfor the lung antigen L762P.

SEQ ID NO:441 is the amino acid sequence of L763 peptide #2684.

SEQ ID NO:442 is the predicted full-length cDNA for the cloned partialsequence of clone L5295 (SEQ ID NO:106).

SEQ ID NO:443 is the deduced amino acid sequence encoded by SEQ IDNO:442.

SEQ ID NO:444 is the forward primer PDM-734 for the coding region ofclone L523S.

SEQ ID NO:445 is the reverse primer PDM-735 for the coding region ofclone L523S.

SEQ ID NO:446 is the amino acid sequence for the expressed recombinantL523S.

SEQ ID NO:447 is the DNA coding sequence for the recombinant L523S.

SEQ ID NO:448 is another forward primer PDM-733 for the coding region ofclone L523S.

SEQ ID NO:449 is the amino acid sequence for a second expressedrecombinant L523S.

SEQ ID NO:450 is the DNA coding sequence for a second recombinant L523S.

SEQ ID NO:451 corresponds to amino acids 86-110, an epitope ofL514S-specific in the generation of antibodies.

SEQ ID NO:452 corresponds to amino acids 21-45, an epitope ofL514S-specific in the generation of antibodies.

SEQ ID NO:453 corresponds to amino acids 121-135, an epitope ofL514S-specific in the generation of antibodies.

SEQ ID NO:454 corresponds to amino acids 440-460, an epitope ofL523S-specific in the generation of antibodies.

SEQ ID NO:455 corresponds to amino acids 156-175, an epitope ofL523S-specific in the generation of antibodies.

SEQ ID NO:456 corresponds to amino acids 326-345, an epitope ofL523S-specific in the generation of antibodies.

SEQ ID NO:457 corresponds to amino acids 40-59, an epitope of

L523S-specific in the generation of antibodies.

SEQ ID NO:458 corresponds to amino acids 80-99, an epitope ofL523S-specific in the generation of antibodies.

SEQ ID NO:459 corresponds to amino acids 160-179, an epitope ofL523S-specific in the generation of antibodies.

SEQ ID NO:460 corresponds to amino acids 180-199, an epitope ofL523S-specific in the generation of antibodies.

SEQ ID NO:461 corresponds to amino acids 320-339, an epitope ofL523S-specific in the generation of antibodies.

SEQ ID NO:462 corresponds to amino acids 340-359, an epitope ofL523S-specific in the generation of antibodies.

SEQ ID NO:463 corresponds to amino acids 370-389, an epitope ofL523S-specific in the generation of antibodies.

SEQ ID NO:464 corresponds to amino acids 380-399, an epitope ofL523S-specific in the generation of antibodies.

SEQ ID NO:465 corresponds to amino acids 37-55, an epitope ofL523S-recognized by the L523S-specific CTL line 6B1.

SEQ ID NO:466 corresponds to amino acids 41-51, the mapped antigenicepitope of L523S-recognized by the L523S-specific CTL line 6B1.

SEQ ID NO:467 corresponds to the DNA sequence which encodes SEQ IDNO:466.

SEQ ID NO:468 corresponds to the amino acids of peptide 16, 17 ofhL523S.

SEQ ID NO:469 corresponds to the amino acids of peptide 16, 17 ofmL523S.

SEQ ID NO:470 corresponds to the amino acids of the 20-mer peptide #4 ofL523S.

SEQ ID NO:471 corresponds to the amino acids of the overlapping 20-merpeptides #14-#19 of L523S.

SEQ ID NO:472 corresponds to the amino acids of the overlapping 20-merpeptides #20-#25 of L523S.

SEQ ID NO:473 corresponds to the amino acids of the overlapping 20-merpeptides #26-#30.5 of L523S.

SEQ ID NO:474 corresponds to the amino acids of the overlapping 20-merpeptides #31-#36 of L523S.

SEQ ID NO:475 corresponds to the amino acids of the overlapping 20-merpeptides #37-#40.5 of L523S.

SEQ ID NO:476 corresponds to the amino acids of the overlapping 20-merpeptides #41-#46.5 of L523S.

SEQ ID NO:477 corresponds to the amino acids of the overlapping 20-merpeptides #47-#53 of L523S.

SEQ ID NO:478 is the cDNA encoding the full length ORF of L523S.

SEQ ID NO:479 is the cDNA sequence of Adenovirus-L523s, an Adenovirusvector containing the cDNA encoding the full-length ORF of L523S.

SEQ ID NO:480 is the amino acid sequence of the full-length L523Sprotein as expressed from the Adenovirus vector set forth in SEQ IDNO:479.

SEQ ID NO:481 is amino acids 9-27 of L523S containing a CD8 T cellepitope as described in example 37.

SEQ ID NO:482 is amino acids 33-75 of L523S containing a CD4 T cellepitope as described in example 37.

SEQ ID NO:483 is the determined cDNA sequence for the Rhesus macaqueL523S homologue.

SEQ ID NO:484 is the predicted amino acid sequence for the Rhesusmacaque L523S homologue, encoded by the polynucleotide sequence setforth in SEQ ID NO:483.

SEQ ID NO:485 is the full-length L523S cDNA, together with its Kozakconsensus sequence and a C-terminal 10×His Tag for expression in insectcells using a baculovirus system.

SEQ ID NO:486 is the full-length L523S amino acid sequence encoded bythe polynucleotide set forth in SEQ ID NO:485.

SEQ ID NO:487 is the L523F1 PCR primer.

SEQ ID NO:488 is the L523RV1 PCR primer.

SEQ ID NO:489 is the cDNA encoding the minimal epitope of L514S setforth in SEQ ID NO:490.

SEQ ID NO:490 is the amino acid sequence of peptide #10 minimal epitopeof L514S.

SEQ ID NO:491 is a minimal 9-mer CTL epitope of L523S.

SEQ ID NO:492 is the amino acid sequence of peptide #2 of NY-ESO-1.

SEQ ID NO:493 is the amino acid sequence of peptide #3 of NY-ESO-1.

SEQ ID NO:494 is the amino acid sequence of peptide #10 of NY-ESO-1.

SEQ ID NO:495 is the amino acid sequence of peptide #17 of NY-ESO-1.

SEQ ID NO:496 is the amino acid sequence of peptide #5 of NY-ESO-1.

SEQ ID NO:497 is the amino acid sequence of peptide #42 of L523S.

SEQ ID NO:498 is the amino acid sequence of IMP-1 peptide #42.

SEQ ID NO:499 is the amino acid sequence of IMP-2 peptide #42.

SEQ ID NO:500 is the amino acid sequence of IMP-1.

SEQ ID NO:501 is the amino acid sequence of IMP-2.

SEQ ID NO:502 is the amino acid sequence of IMP-1 peptide #32.

SEQ ID NO:503 is the amino acid sequence of IMP-2 peptide #32.

SEQ ID NO:504 is the amino acid sequence of peptide #1 of L523S.

SEQ ID NO:505 is the amino acid sequence of peptide #2 of L523S.

SEQ ID NO:506 is the amino acid sequence of peptide #3 of L523S.

SEQ ID NO:507 is the amino acid sequence of peptide #4 of L523S.

SEQ ID NO:508 is the amino acid sequence of peptide #5 of L523S.

SEQ ID NO:509 is the amino acid sequence of peptide #6 of L523S.

SEQ ID NO:510 is the amino acid sequence of peptide #7 of L523S.

SEQ ID NO:511 is the amino acid sequence of peptide #8 of L523S.

SEQ ID NO:512 is the amino acid sequence of peptide #9 of L523S.

SEQ ID NO:513 is the amino acid sequence of peptide #10 of L523S.

SEQ ID NO:514 is the amino acid sequence of peptide #11 of L523S.

SEQ ID NO:515 is the amino acid sequence of peptide #12 of L523S.

SEQ ID NO:516 is the amino acid sequence of peptide #13 of L523S.

SEQ ID NO:517 is the amino acid sequence of peptide #14 of L523S.

SEQ ID NO:518 is the amino acid sequence of peptide #15 of L523S.

SEQ ID NO:519 is the amino acid sequence of peptide #16 of L523S.

SEQ ID NO:520 is the amino acid sequence of peptide #17 of L523S.

SEQ ID NO:521 is the amino acid sequence of peptide #18 of L523S.

SEQ ID NO:522 is the amino acid sequence of peptide #19 of L523S.

SEQ ID NO:523 is the amino acid sequence of peptide #20 of L523S.

SEQ ID NO:524 is the amino acid sequence of peptide #21 of L523S.

SEQ ID NO:525 is the amino acid sequence of peptide #22 of L523S.

SEQ ID NO:526 is the amino acid sequence of peptide #23 of L523S.

SEQ ID NO:527 is the amino acid sequence of peptide #24 of L523S.

SEQ ID NO:528 is the amino acid sequence of peptide #25 of L523S.

SEQ ID NO:529 is the amino acid sequence of peptide #26 of L523S.

SEQ ID NO:530 is the amino acid sequence of peptide #27 of L523S.

SEQ ID NO:531 is the amino acid sequence of peptide #28 of L523S.

SEQ ID NO:532 is the amino acid sequence of peptide #29 of L523S.

SEQ ID NO:533 is the amino acid sequence of peptide #30 of L523S.

SEQ ID NO:534 is the amino acid sequence of peptide #30.5 of L523S.

SEQ ID NO:535 is the amino acid sequence of peptide #31 of L523S.

SEQ ID NO:536 is the amino acid sequence of peptide #32 of L523S.

SEQ ID NO:537 is the amino acid sequence of peptide #33 of L523S.

SEQ ID NO:538 is the amino acid sequence of peptide #34 of L523S.

SEQ ID NO:539 is the amino acid sequence of peptide #35 of L523S.

SEQ ID NO:540 is the amino acid sequence of peptide #36 of L523S.

SEQ ID NO:541 is the amino acid sequence of peptide #37 of L523S.

SEQ ID NO:542 is the amino acid sequence of peptide #38 of L523S.

SEQ ID NO:543 is the amino acid sequence of peptide #38.5 of L523S.

SEQ ID NO:544 is the amino acid sequence of peptide #39 of L523S.

SEQ ID NO:545 is the amino acid sequence of peptide #40 of L523S.

SEQ ID NO:546 is the amino acid sequence of peptide #40.5 of L523S.

SEQ ID NO:547 is the amino acid sequence of peptide #41 of L523S.

SEQ ID NO:548 is the amino acid sequence of peptide #42 of L523S.

SEQ ID NO:549 is the amino acid sequence of peptide #43 of L523S.

SEQ ID NO:550 is the amino acid sequence of peptide #44 of L523S.

SEQ ID NO:551 is the amino acid sequence of peptide #45 of L523S.

SEQ ID NO:552 is the amino acid sequence of peptide #46 of L523S.

SEQ ID NO:553 is the amino acid sequence of peptide #46.5 of L523S.

SEQ ID NO:554 is the amino acid sequence of peptide #47 of L523S.

SEQ ID NO:555 is the amino acid sequence of peptide #48 of L523S.

SEQ ID NO:556 is the amino acid sequence of peptide #49 of L523S.

SEQ ID NO:557 is the amino acid sequence of peptide #50 of L523S.

SEQ ID NO:558 is the amino acid sequence of peptide #51 of L523S.

SEQ ID NO:559 is the amino acid sequence of peptide #52 of L523S.

SEQ ID NO:560 is the amino acid sequence of peptide #53 of L523S.

SEQ ID NO: 561 is the amino acid sequence of the mouse ortholog ofL762P.

SEQ ID NO: 562 is the amino acid sequence of a peptide recognized bymouse monoclonal antibodies 153A12 and 153A20.

SEQ ID NO: 563 is the amino acid sequence of a peptide recognized byhuman monoclonal antibody 2.4.1.

DETAILED DESCRIPTION OF THE INVENTION

U.S. patents, U.S. patent application publications, U.S. patentapplications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, are incorporated herein by reference, intheir entirety.

The present invention is directed generally to compositions and theiruse in the therapy and diagnosis of cancer, particularly lung cancer. Asdescribed further below, illustrative compositions of the presentinvention include, but are not restricted to, polypeptides, particularlyimmunogenic polypeptides, polynucleotides encoding such polypeptides,antibodies and other binding agents, antigen presenting cells (APCs) andimmune system cells (e.g., T cells).

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al. Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Polypeptide Compositions

As used herein, the term “polypeptide” is used in its conventionalmeaning, i.e., as a sequence of amino acids. The polypeptides are notlimited to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide, and such terms may be used interchangeably herein unlessspecifically indicated otherwise. This term also does not refer to orexclude post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising epitopes, i.e.,antigenic determinants substantially responsible for the immunogenicproperties of a polypeptide and being capable of evoking an immuneresponse.

Particularly illustrative polypeptides of the present invention comprisethose encoded by a polynucleotide sequence set forth in any one of SEQID NO:1-3, 6-8, 10-13, 15-27, 29, 30, 32, 34-49, 51, 52, 54, 55, 57-59,61-69, 71, 73, 74, 77, 78, 80-82, 84, 86-96, 107-109, 111, 113, 125,127, 128, 129, 131-133, 142, 144, 148-151, 153, 154, 157, 158, 160, 167,168, 171, 179, 182, 184-186, 188-191, 193, 194, 198-207, 209, 210, 213,214, 217, 220-224, 253-337, 345, 347, 349, 358, 362, 364, 365, 368,370-375, 420, 424, 428, 431, 434, 442, 447, 450, 467, 478, 479 and 483,or a sequence that hybridizes under moderately stringent conditions, or,alternatively, under highly stringent conditions, to a polynucleotidesequence set forth in any one of SEQ ID NO:1-3, 6-8, 10-13, 15-27, 29,30, 32, 34-49, 51, 52, 54, 55, 57-59, 61-69, 71, 73, 74, 77, 78, 80-82,84, 86-96, 107-109, 111, 113, 125, 127, 128, 129, 131-133, 142, 144,148-151, 153, 154, 157, 158, 160, 167, 168, 171, 179, 182, 184-186,188-191, 193, 194, 198-207, 209, 210, 213, 214, 217, 220-224, 253-337,345, 347, 349, 358, 362, 364, 365, 368, 370-375, 420, 424, 428, 431,434, 442, 447, 450, 467, 478, 479 and 483. Certain illustrativepolypeptides of the invention comprise amino acid sequences as set forthin any one of SEQ ID NO:152, 155, 156, 165, 166, 169, 170, 172, 174,176, 226-252, 338-344, 346, 350, 357, 361, 363, 365, 367, 369, 376-382,387-419, 423, 427, 430, 433, 441, 443, 446, 449, 451-466, 468-477,480-482, and 484.

The polypeptides of the present invention are sometimes herein referredto as lung tumor proteins or lung tumor polypeptides, as an indicationthat their identification has been based at least in part upon theirincreased levels of expression in lung tumor samples. Thus, a “lungtumor polypeptide” or “lung tumor protein,” refers generally to apolypeptide sequence of the present invention, or a polynucleotidesequence encoding such a polypeptide, that is expressed in a substantialproportion of lung tumor samples, for example preferably greater thanabout 20%, more preferably greater than about 30%, and most preferablygreater than about 50% or more of lung tumor samples tested, at a levelthat is at least two fold, and preferably at least five fold, greaterthan the level of expression in normal tissues, as determined using arepresentative assay provided herein. A lung tumor polypeptide sequenceof the invention, based upon its increased level of expression in tumorcells, has particular utility both as a diagnostic marker as well as atherapeutic target, as further described below.

In certain preferred embodiments, the polypeptides of the invention areimmunogenic, i.e., they react detectably within an immunoassay (such asan ELISA or T-cell stimulation assay) with antisera and/or T-cells froma patient with lung cancer. Screening for immunogenic activity can beperformed using techniques well known to the skilled artisan. Forexample, such screens can be performed using methods such as thosedescribed in Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988. In one illustrative example, apolypeptide may be immobilized on a solid support and contacted withpatient sera to allow binding of antibodies within the sera to theimmobilized polypeptide. Unbound sera may then be removed and boundantibodies detected using, for example, ¹²⁵1-labeled Protein A.

As would be recognized by the skilled artisan, immunogenic portions ofthe polypeptides disclosed herein are also encompassed by the presentinvention. An “immunogenic portion,” as used herein, is a fragment of animmunogenic polypeptide of the invention that itself is immunologicallyreactive (i.e., specifically binds) with the B-cells and/or T-cellsurface antigen receptors that recognize the polypeptide. Immunogenicportions may generally be identified using well known techniques, suchas those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247(Raven Press, 1993) and references cited therein. Such techniquesinclude screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well-known techniques.

In one preferred embodiment, an immunogenic portion of a polypeptide ofthe present invention is a portion that reacts with antisera and/orT-cells at a level that is not substantially less than the reactivity ofthe full-length polypeptide (e.g., in an ELISA and/or T-cell reactivityassay). Preferably, the level of immunogenic activity of the immunogenicportion is at least about 50%, preferably at least about 70% and mostpreferably greater than about 90% of the immunogenicity for thefull-length polypeptide. In some instances, preferred immunogenicportions will be identified that have a level of immunogenic activitygreater than that of the corresponding full-length polypeptide, e.g.,having greater than about 100% or 150% or more immunogenic activity.

In certain other embodiments, illustrative immunogenic portions mayinclude peptides in which an N-terminal leader sequence and/ortransmembrane domain have been deleted. Other illustrative immunogenicportions will contain a small N- and/or C-terminal deletion (e.g., 1-30amino acids, preferably 5-15 amino acids), relative to the matureprotein.

In another embodiment, a polypeptide composition of the invention mayalso comprise one or more polypeptides that are immunologically reactivewith T cells and/or antibodies generated against a polypeptide of theinvention, particularly a polypeptide having an amino acid sequencedisclosed herein, or to an immunogenic fragment or variant thereof.

In another embodiment of the invention, polypeptides are provided thatcomprise one or more polypeptides that are capable of eliciting T cellsand/or antibodies that are immunologically reactive with one or morepolypeptides described herein, or one or more polypeptides encoded bycontiguous nucleic acid sequences contained in the polynucleotidesequences disclosed herein, or immunogenic fragments or variantsthereof, or to one or more nucleic acid sequences which hybridize to oneor more of these sequences under conditions of moderate to highstringency.

The present invention, in another aspect, provides polypeptide fragmentscomprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous aminoacids, or more, including all intermediate lengths, of a polypeptidecompositions set forth herein, such as those set forth in SEQ ID NO:152,155, 156, 165, 166, 169, 170, 172, 174, 176, 226-252, 338-344, 346, 350,357, 361, 363, 365, 367, 369, 376-382 and 387-419, 441, 443, 446, 449,451-466, 468-477, 480-482, and 484, or those encoded by a polynucleotidesequence set forth in a sequence of SEQ ID NO:1-3, 6-8, 10-13, 15-27,29, 30, 32, 34-49, 51, 52, 54, 55, 57-59, 61-69, 71, 73, 74, 77, 78,80-82, 84, 86-96, 107-109, 111, 113, 125, 127, 128, 129, 131-133, 142,144, 148-151, 153, 154, 157, 158, 160, 167, 168, 171, 179, 182, 184-186,188-191, 193, 194, 198-207, 209, 210, 213, 214, 217, 220-224, 253-337,345, 347, 349, 358, 362, 364, 365, 368, 370-375, 420, 424, 428, 431,434, 442, 447, 450, 467, 478, 479 and 483.

In another aspect, the present invention provides variants of thepolypeptide compositions described herein. Polypeptide variantsgenerally encompassed by the present invention will typically exhibit atleast about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more identity (determined as described below), along itslength, to a polypeptide sequences set forth herein.

In one preferred embodiment, the polypeptide fragments and variantsprovide by the present invention are immunologically reactive with anantibody and/or T-cell that reacts with a full-length polypeptidespecifically set for the herein.

In another preferred embodiment, the polypeptide fragments and variantsprovided by the present invention exhibit a level of immunogenicactivity of at least about 50%, preferably at least about 70%, and mostpreferably at least about 90% or more of that exhibited by a full-lengthpolypeptide sequence specifically set forth herein.

A polypeptide “variant,” as the term is used herein, is a polypeptidethat typically differs from a polypeptide specifically disclosed hereinin one or more substitutions, deletions, additions and/or insertions.Such variants may be naturally occurring or may be syntheticallygenerated, for example, by modifying one or more of the abovepolypeptide sequences of the invention and evaluating their immunogenicactivity as described herein and/or using any of a number of techniqueswell known in the art.

For example, certain illustrative variants of the polypeptides of theinvention include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other illustrative variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

In many instances, a variant will contain conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. As described above, modifications may be made in thestructure of the polynucleotides and polypeptides of the presentinvention and still obtain a functional molecule that encodes a variantor derivative polypeptide with desirable characteristics, e.g., withimmunogenic characteristics. When it is desired to alter the amino acidsequence of a polypeptide to create an equivalent, or even an improved,immunogenic variant or portion of a polypeptide of the invention, oneskilled in the art will typically change one or more of the codons ofthe encoding DNA sequence according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said peptides withoutappreciable loss of their biological utility or activity.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(specifically incorporated herein by reference in its entirety), statesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0 ±1); glutamate (+3.0 ±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (31 3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

In addition, any polynucleotide may be further modified to increasestability in vivo. Possible modifications include, but are not limitedto, the addition of flanking sequences at the 5′ and/or 3′ ends; the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl- methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain non-conservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe immunogenicity, secondary structure and hydropathic nature of thepolypeptide.

As noted above, polypeptides may comprise a signal (or leader) sequenceat the N-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

When comparing polypeptide sequences, two sequences are said to be“identical” if the sequence of amino acids in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides and polypeptides of the invention.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. For amino acid sequences,a scoring matrix can be used to calculate the cumulative score.Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

In one preferred approach, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e., the window size) and multiplying the results by 100 toyield the percentage of sequence identity.

Within other illustrative embodiments, a polypeptide may be a fusionpolypeptide that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence, such as a known tumor protein. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the polypeptide or to enable the polypeptide to betargeted to desired intracellular compartments. Still further fusionpartners include affinity tags, which facilitate purification of thepolypeptide.

Fusion polypeptides may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion polypeptide isexpressed as a recombinant polypeptide, allowing the production ofincreased levels, relative to a non-fused polypeptide, in an expressionsystem. Briefly, DNA sequences encoding the polypeptide components maybe assembled separately, and ligated into an appropriate expressionvector. The 3′ end of the DNA sequence encoding one polypeptidecomponent is ligated, with or without a peptide linker, to the 5′ end ofa DNA sequence encoding the second polypeptide component so that thereading frames of the sequences are in phase. This permits translationinto a single fusion polypeptide that retains the biological activity ofboth component polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion polypeptideusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

The fusion polypeptide can comprise a polypeptide as described hereintogether with an unrelated immunogenic protein, such as an immunogenicprotein capable of eliciting a recall response. Examples of suchproteins include tetanus, tuberculosis and hepatitis proteins (see, forexample, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

In one preferred embodiment, the immunological fusion partner is derivedfrom a Mycobacterium sp., such as a Mycobacterium tuberculosis-derivedRa12 fragment. Ra12 compositions and methods for their use in enhancingthe expression and/or immunogenicity of heterologouspolynucleotide/polypeptide sequences is described in U.S. PatentApplication 60/158,585, the disclosure of which is incorporated hereinby reference in its entirety. Briefly, Ra12 refers to a polynucleotideregion that is a subsequence of a Mycobacterium tuberculosis MTB32Anucleic acid. MTB32A is a serine protease of 32 KD molecular weightencoded by a gene in virulent and avirulent strains of M. tuberculosis.The nucleotide sequence and amino acid sequence of MTB32A have beendescribed (for example, U.S. Patent Application 60/158,585; see also,Skeiky et al., Infection and Immun. (1999) 67:3998-4007, incorporatedherein by reference). C-terminal fragments of the MTB32A coding sequenceexpress at high levels and remain as a soluble polypeptides throughoutthe purification process. Moreover, Ra12 may enhance the immunogenicityof heterologous immunogenic polypeptides with which it is fused. Onepreferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragmentcorresponding to amino acid residues 192 to 323 of MTB32A.

Other preferred Ra12 polynucleotides generally comprise at least about15 consecutive nucleotides, at least about 30 nucleotides, at leastabout 60 nucleotides, at least about 100 nucleotides, at least about 200nucleotides, or at least about 300 nucleotides that encode a portion ofa Ra12 polypeptide.

Ra12 polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a Ra12 polypeptide or a portion thereof) or maycomprise a variant of such a sequence. Ra12 polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the biological activity of the encoded fusionpolypeptide is not substantially diminished, relative to a fusionpolypeptide comprising a native Ra12 polypeptide. Variants preferablyexhibit at least about 70% identity, more preferably at least about 80%identity and most preferably at least about 90% identity to apolynucleotide sequence that encodes a native Ra12 polypeptide or aportion thereof.

Within other preferred embodiments, an immunological fusion partner isderived from protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenza virus, NS1 (haemagglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionpolypeptide. A repeat portion is found in the C-terminal region startingat residue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

Yet another illustrative embodiment involves fusion polypeptides, andthe polynucleotides encoding them, wherein the fusion partner comprisesa targeting signal capable of directing a polypeptide to theendosomal/lysosomal compartment, as described in U.S. Pat. No.5,633,234. An immunogenic polypeptide of the invention, when fused withthis targeting signal, will associate more efficiently with MHC class IImolecules and thereby provide enhanced in vivo stimulation of CD4⁺T-cells specific for the polypeptide.

Polypeptides of the invention are prepared using any of a variety ofwell known synthetic and/or recombinant techniques, the latter of whichare further described below. Polypeptides, portions and other variantsgenerally less than about 150 amino acids can be generated by syntheticmeans, using techniques well known to those of ordinary skill in theart. In one illustrative example, such polypeptides are synthesizedusing any of the commercially available solid-phase techniques, such asthe Merrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain. See Merrifield, J. Am.Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

In general, polypeptide compositions (including fusion polypeptides) ofthe invention are isolated. An “isolated” polypeptide is one that isremoved from its original environment. For example, anaturally-occurring protein or polypeptide is isolated if it isseparated from some or all of the coexisting materials in the naturalsystem. Preferably, such polypeptides are also purified, e.g., are atleast about 90% pure, more preferably at least about 95% pure and mostpreferably at least about 99% pure.

Polynucleotide Compositions

The present invention, in other aspects, provides polynucleotidecompositions. The terms “DNA” and “polynucleotide” are used essentiallyinterchangeably herein to refer to a DNA molecule that has been isolatedfree of total genomic DNA of a particular species. “Isolated,” as usedherein, means that a polynucleotide is substantially away from othercoding sequences, and that the DNA molecule does not contain largeportions of unrelated coding DNA, such as large chromosomal fragments orother functional genes or polypeptide coding regions. Of course, thisrefers to the DNA molecule as originally isolated, and does not excludegenes or coding regions later added to the segment by the hand of man.

As will be understood by those skilled in the art, the polynucleotidecompositions of this invention can include genomic sequences,extra-genomic and plasmid-encoded sequences and smaller engineered genesegments that express, or may be adapted to express, proteins,polypeptides, peptides and the like. Such segments may be naturallyisolated, or modified synthetically by the hand of man.

As will be also recognized by the skilled artisan, polynucleotides ofthe invention may be single-stranded (coding or antisense) ordouble-stranded, and may be DNA (genomic, cDNA or synthetic) or RNAmolecules. RNA molecules may include HnRNA molecules, which containintrons and correspond to a DNA molecule in a one-to-one manner, andmRNA molecules, which do not contain introns. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a polypeptide/protein of the invention or aportion thereof) or may comprise a sequence that encodes a variant orderivative, preferably and immunogenic variant or derivative, of such asequence.

Therefore, according to another aspect of the present invention,polynucleotide compositions are provided that comprise some or all of apolynucleotide sequence set forth in any one of SEQ ID NO:1-3, 6-8,10-13, 15-27, 29, 30, 32, 34-49, 51, 52, 54, 55, 57-59, 61-69, 71, 73,74, 77, 78, 80-82, 84, 86-96, 107-109, 111, 113, 125, 127, 128, 129,131-133, 142, 144, 148-151, 153, 154, 157, 158, 160, 167, 168, 171, 179,182, 184-186, 188-191, 193, 194, 198-207, 209, 210, 213, 214, 217,220-224, 253-337, 345, 347, 349, 358, 362, 364, 365, 368, 370-375, 420,424, 428, 431, 434, 442, 447, 450, 467, 478, 479, 483, 485, and 489,complements of a polynucleotide sequence set forth in any one of SEQ IDNO:1-3, 6-8, 10-13, 15-27, 29, 30, 32, 34-49, 51, 52, 54, 55, 57-59,61-69, 71, 73, 74, 77, 78, 80-82, 84, 86-96, 107-109, 111, 113, 125,127, 128, 129, 131-133, 142, 144, 148-151, 153, 154, 157, 158, 160, 167,168, 171, 179, 182, 184-186, 188-191, 193, 194, 198-207, 209, 210, 213,214, 217, 220-224, 253-337, 345, 347, 349, 358, 362, 364, 365, 368,370-375, 420, 424, 428, 431, 434, 442, 447, 450, 467, 478, 479, 483,485, and 489, and degenerate variants of a polynucleotide sequence setforth in any one of SEQ ID NO:1-3, 6-8, 10-13, 15-27, 29, 30, 32, 34-49,51, 52, 54, 55, 57-59, 61-69, 71, 73, 74, 77, 78, 80-82, 84, 86-96,107-109, 111, 113, 125, 127, 128, 129, 131-133, 142, 144, 148-151, 153,154, 157, 158, 160, 167, 168, 171, 179, 182, 184-186, 188-191, 193, 194,198-207, 209, 210, 213, 214, 217, 220-224, 253-337, 345, 347, 349, 358,362, 364, 365, 368, 370-375, 420, 424, 428, 431, 434, 442, 447, 450,467, 478, 479, 483, 485, and 489. In certain preferred embodiments, thepolynucleotide sequences set forth herein encode immunogenicpolypeptides, as described above.

In other related embodiments, the present invention providespolynucleotide variants having substantial identity to the sequencesdisclosed herein in SEQ ID NO:1-3, 6-8, 10-13, 15-27, 29, 30, 32, 34-49,51, 52, 54, 55, 57-59, 61-69, 71, 73, 74, 77, 78, 80-82, 84, 86-96,107-109, 111, 113, 125, 127, 128, 129, 131-133, 142, 144, 148-151, 153,154, 157, 158, 160, 167, 168, 171, 179, 182, 184-186, 188-191, 193, 194,198-207, 209, 210, 213, 214, 217, 220-224, 253-337, 345, 347, 349, 358,362, 364, 365, 368, 370-375, 420, 424, 428, 431, 434, 442, 447, 450,467, 478, 479, 483, 485, and 489, for example those comprising at least70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% or higher, sequence identity compared to apolynucleotide sequence of this invention using the methods describedherein, (e.g., BLAST analysis using standard parameters, as describedbelow). One skilled in this art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning and thelike.

Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the immunogenicity of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein). Theterm “variants” should also be understood to encompasses homologousgenes of xenogenic origin.

In additional embodiments, the present invention provides polynucleotidefragments comprising various lengths of contiguous stretches of sequenceidentical to or complementary to one or more of the sequences disclosedherein. For example, polynucleotides are provided by this invention thatcomprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300,400, 500 or 1000 or more contiguous nucleotides of one or more of thesequences disclosed herein as well as all intermediate lengths therebetween. It will be readily understood that “intermediate lengths”, inthis context, means any length between the quoted values, such as 16,17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53,etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including allintegers through 200-500; 500-1,000, and the like.

In another embodiment of the invention, polynucleotide compositions areprovided that are capable of hybridizing under moderate to highstringency conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5× SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2×SSC containing 0.1% SDS. One skilled in the art will understandthat the stringency of hybridization can be readily manipulated, such asby altering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, e.g., to 60-65° C. or 65-70° C.

In certain preferred embodiments, the polynucleotides described above,e.g., polynucleotide variants, fragments and hybridizing sequences,encode polypeptides that are immunologically cross-reactive with apolypeptide sequence specifically set forth herein. In other preferredembodiments, such polynucleotides encode polypeptides that have a levelof immunogenic activity of at least about 50%, preferably at least about70%, and more preferably at least about 90% of that for a polypeptidesequence specifically set forth herein.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, illustrative polynucleotidesegments with total lengths of about 10,000, about 5000, about 3000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

When comparing polynucleotide sequences, two sequences are said to be“identical” if the sequence of nucleotides in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Optimal alignment of sequences forcomparison may be conducted using the Megalign program in the Lasergenesuite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), usingdefault parameters. This program embodies several alignment schemesdescribed in the following references: Dayhoff, M. O. (1978) A model ofevolutionary change in proteins—Matrices for detecting distantrelationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence andStructure, National Biomedical Research Foundation, Washington D.C. Vol.5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignmentand Phylogenes pp. 626-645 Methods in Enzymology vol. 183, AcademicPress, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989)CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11-17;Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987)Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973)Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy,Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J.(1983) Proc. Natl. Acad., Sci. USA 80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides of the invention. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12percent, as compared to the reference sequences (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid bases occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the reference sequence (i.e., thewindow size) and multiplying the results by 100 to yield the percentageof sequence identity.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene.

Nonetheless, polynucleotides that vary due to differences in codon usageare specifically contemplated by the present invention. Further, allelesof the genes comprising the polynucleotide sequences provided herein arewithin the scope of the present invention. Alleles are endogenous genesthat are altered as a result of one or more mutations, such asdeletions, additions and/or substitutions of nucleotides. The resultingmRNA and protein may, but need not, have an altered structure orfunction. Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, is employed for thepreparation of immunogenic variants and/or derivatives of thepolypeptides described herein. By this approach, specific modificationsin a polypeptide sequence can be made through mutagenesis of theunderlying polynucleotides that encode them. These techniques provides astraightforward approach to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into thepolynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theimmunogenicity of a polypeptide vaccine. The techniques of site-specificmutagenesis are well-known in the art, and are widely used to createvariants of both polypeptides and polynucleotides. For example,site-specific mutagenesis is often used to alter a specific portion of aDNA molecule. In such embodiments, a primer comprising typically about14 to about 25 nucleotides or so in length is employed, with about 5 toabout 10 residues on both sides of the junction of the sequence beingaltered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage are readily commercially-available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

In another approach for the production of polypeptide variants of thepresent invention, recursive sequence recombination, as described inU.S. Pat. No. 5,837,458, may be employed. In this approach, iterativecycles of recombination and screening or selection are performed to“evolve” individual polynucleotide variants of the invention having, forexample, enhanced immunogenic activity.

In other embodiments of the present invention, the polynucleotidesequences provided herein can be advantageously used as probes orprimers for nucleic acid hybridization. As such, it is contemplated thatnucleic acid segments that comprise a sequence region of at least about15 nucleotide long contiguous sequence that has the same sequence as, oris complementary to, a 15 nucleotide long contiguous sequence disclosedherein will find particular utility. Longer contiguous identical orcomplementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200,500, 1000 (including all intermediate lengths) and even up to fulllength sequences will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to asequence of interest will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are also envisioned, such as the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so (including intermediate lengths as well),identical or complementary to a polynucleotide sequence disclosedherein, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting. This would allow a geneproduct, or fragment thereof, to be analyzed, both in diverse cell typesand also in various bacterial cells. The total size of fragment, as wellas the size of the complementary stretch(es), will ultimately depend onthe intended use or application of the particular nucleic acid segment.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 15 and about 100 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

The use of a hybridization probe of about 15-25 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 15 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 25 contiguous nucleotides,or even longer where desired.

Hybridization probes may be selected from any portion of any of thesequences disclosed herein. All that is required is to review thesequences set forth herein, or to any continuous portion of thesequences, from about 15-25 nucleotides in length up to and includingthe full length sequence, that one wishes to utilize as a probe orprimer. The choice of probe and primer sequences may be governed byvarious factors. For example, one may wish to employ primers fromtowards the termini of the total sequence.

Small polynucleotide segments or fragments may be readily prepared by,for example, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. No. 4,683,202(incorporated herein by reference), by introducing selected sequencesinto recombinant vectors for recombinant production, and by otherrecombinant DNA techniques generally known to those of skill in the artof molecular biology.

The nucleotide sequences of the invention may be used for their abilityto selectively form duplex molecules with complementary stretches of theentire gene or gene fragments of interest. Depending on the applicationenvisioned, one will typically desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by a salt concentration of fromabout 0.02 M to about 0.15 M salt at temperatures of from about 50° C.to about 70° C. Such selective conditions tolerate little, if any,mismatch between the probe and the template or target strand, and wouldbe particularly suitable for isolating related sequences.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent (reduced stringency) hybridizationconditions will typically be needed in order to allow formation of theheteroduplex. In these circumstances, one may desire to employ saltconditions such as those of from about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Cross-hybridizingspecies can thereby be readily identified as positively hybridizingsignals with respect to control hybridizations. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

According to another embodiment of the present invention, polynucleotidecompositions comprising antisense oligonucleotides are provided.Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, provide atherapeutic approach by which a disease can be treated by inhibiting thesynthesis of proteins that contribute to the disease. The efficacy ofantisense oligonucleotides for inhibiting protein synthesis is wellestablished. For example, the synthesis of polygalactauronase and themuscarine type 2 acetylcholine receptor are inhibited by antisenseoligonucleotides directed to their respective mRNA sequences (U.S. Pat.No. 5,739,119 and U.S. Pat. No. 5,759,829). Further, examples ofantisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin,STK-1, striatal GABA_(A) receptor and human EGF (Jaskulski et al.,Science. 1988 Jun. 10; 240(4858):1544-6; Vasanthakumar and Ahmed, CancerCommun. 1989;1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998Jun. 15; 57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573;U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). Antisenseconstructs have also been described that inhibit and can be used totreat a variety of abnormal cellular proliferations, e.g. cancer (U.S.Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No.5,783,683).

Therefore, in certain embodiments, the present invention providesoligonucleotide sequences that comprise all, or a portion of, anysequence that is capable of specifically binding to polynucleotidesequence described herein, or a complement thereof. In one embodiment,the antisense oligonucleotides comprise DNA or derivatives thereof. Inanother embodiment, the oligonucleotides comprise RNA or derivativesthereof. In a third embodiment, the oligonucleotides are modified DNAscomprising a phosphorothioated modified backbone. In a fourthembodiment, the oligonucleotide sequences comprise peptide nucleic acidsor derivatives thereof. In each case, preferred compositions comprise asequence region that is complementary, and more preferablysubstantially-complementary, and even more preferably, completelycomplementary to one or more portions of polynucleotides disclosedherein. Selection of antisense compositions specific for a given genesequence is based upon analysis of the chosen target sequence anddetermination of secondary structure, T_(m), binding energy, andrelative stability. Antisense compositions may be selected based upontheir relative inability to form dimers, hairpins, or other secondarystructures that would reduce or prohibit specific binding to the targetmRNA in a host cell. Highly preferred target regions of the mRNA, arethose which are at or near the AUG translation initiation codon, andthose sequences which are substantially complementary to 5′ regions ofthe mRNA. These secondary structure analyses and target site selectionconsiderations can be performed, for example, using v.4 of the OLIGOprimer analysis software and/or the BLASTN 2.0.5 algorithm software(Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

The use of an antisense delivery method employing a short peptidevector, termed MPG (27 residues), is also contemplated. The MPG peptidecontains a hydrophobic domain derived from the fusion sequence of HIVgp41 and a hydrophilic domain from the nuclear localization sequence ofSV40 T-antigen (Morris et al., Nucleic Acids Res. 1997 Jul. 15;25(14):2730-6). It has been demonstrated that several molecules of theMPG peptide coat the antisense oligonucleotides and can be deliveredinto cultured mammalian cells in less than 1 hour with relatively highefficiency (90%). Further, the interaction with MPG strongly increasesboth the stability of the oligonucleotide to nuclease and the ability tocross the plasma membrane.

According to another embodiment of the invention, the polynucleotidecompositions described herein are used in the design and preparation ofribozyme molecules for inhibiting expression of the tumor polypeptidesand proteins of the present invention in tumor cells. Ribozymes areRNA-protein complexes that cleave nucleic acids in a site-specificfashion. Ribozymes have specific catalytic domains that possessendonuclease activity (Kim and Cech, Proc Natl Acad Sci U S A. 1987December; 84(24):8788-92; Forster and Symons, Cell. 1987 Apr. 24;49(2):211-20). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Cech et al., Cell. 1981 December; 27(3 Pt 2):487-96; Micheland Westhof, J Mol Biol. 1990 Dec. 5; 216(3):585-610; Reinhold-Hurek andShub, Nature. 1992 May 14; 357(6374):173-6). This specificity has beenattributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofa enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., Proc Natl Acad SciU S A. 1992 Aug. 15; 89(16):7305-9). Thus, the specificity of action ofa ribozyme is greater than that of an antisense oligonucleotide bindingthe same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. NucleicAcids Res. 1992 Sep. 11; 20(17):4559-65. Examples of hairpin motifs aredescribed by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257),Hampel and Tritz, Biochemistry 1989 Jun. 13; 28(12):4929-33; Hampel etal., Nucleic Acids Res. 1990 Jan. 25; 18(2):299-304 and U.S. Pat. No.5,631,359. An example of the hepatitis δ virus motif is described byPerrotta and Been, Biochemistry. 1992 Dec. 1; 31(47):11843-52; anexample of the RNaseP motif is described by Guerrier-Takada et al.,Cell. 1983 December; 35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motifis described by Collins (Saville and Collins, Cell. 1990 May 18;61(4):685-96; Saville and Collins, Proc Natl Acad Sci U S A. 1991 Oct.1; 88(19):8826-30; Collins and Olive, Biochemistry. 1993 Mar. 23;32(11):2795-9); and an example of the Group I intron is described in(U.S. Pat. No. 4,987,071). All that is important in an enzymatic nucleicacid molecule of this invention is that it has a specific substratebinding site which is complementary to one or more of the target geneRNA regions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specificallyincorporated herein by reference) and synthesized to be tested in vitroand in vivo, as described. Such ribozymes can also be optimized fordelivery. While specific examples are provided, those in the art willrecognize that equivalent RNA targets in other species can be utilizedwhen necessary.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No.WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes thegeneral methods for delivery of enzymatic RNA molecules. Ribozymes maybe administered to cells by a variety of methods known to those familiarto the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stint. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569, each specifically incorporated herein byreference.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used, providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells. Ribozymes expressed from suchpromoters have been shown to function in mammalian cells. Suchtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated vectors), or viral RNA vectors (such as retroviral,semliki forest virus, sindbis virus vectors).

In another embodiment of the invention, peptide nucleic acids (PNAs)compositions are provided. PNA is a DNA mimic in which the nucleobasesare attached to a pseudopeptide backbone (Good and Nielsen, AntisenseNucleic Acid Drug Dev. 1997 7(4) 431-37). PNA is able to be utilized ina number methods that traditionally have used RNA or DNA. Often PNAsequences perform better in techniques than the corresponding RNA or DNAsequences and have utilities that are not inherent to RNA or DNA. Areview of PNA including methods of making, characteristics of, andmethods of using, is provided by Corey (Trends Biotechnol 1997 June;15(6):224-9). As such, in certain embodiments, one may prepare PNAsequences that are complementary to one or more portions of the ACE mRNAsequence, and such PNA compositions may be used to regulate, alter,decrease, or reduce the translation of ACE-specific mRNA, and therebyalter the level of ACE activity in a host cell to which such PNAcompositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., Science 1991 Dec. 6;254(5037):1497-500; Hanvey et al., Science. 1992 Nov. 27;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 January;4(1):5-23). This chemistry has three important consequences: firstly, incontrast to DNA or phosphorothioate oligonucleotides, PNAs are neutralmolecules; secondly, PNAs are achiral, which avoids the need to developa stereoselective synthesis; and thirdly, PNA synthesis uses standardBoc or Fmoc protocols for solid-phase peptide synthesis, although othermethods, including a modified Merrifield method, have been used.

PNA monomers or ready-made oligomers are commercially available fromPerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Bocor Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al., Bioorg Med Chem. 1995 April; 3(4):437-45). Themanual protocol lends itself to the production of chemically modifiedPNAs or the simultaneous synthesis of families of closely related PNAs.

As with peptide synthesis, the success of a particular PNA synthesiswill depend on the properties of the chosen sequence. For example, whilein theory PNAs can incorporate any combination of nucleotide bases, thepresence of adjacent purines can lead to deletions of one or moreresidues in the product. In expectation of this difficulty, it issuggested that, in producing PNAs with adjacent purines, one shouldrepeat the coupling of residues likely to be added inefficiently. Thisshould be followed by the purification of PNAs by reverse-phasehigh-pressure liquid chromatography, providing yields and purity ofproduct similar to those observed during the synthesis of peptides.

Modifications of PNAs for a given application may be accomplished bycoupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposed N-terminalamine. Alternatively, PNAs can be modified after synthesis by couplingto an introduced lysine or cysteine. The ease with which PNAs can bemodified facilitates optimization for better solubility or for specificfunctional requirements. Once synthesized, the identity of PNAs andtheir derivatives can be confirmed by mass spectrometry. Several studieshave made and utilized modifications of PNAs (for example, Norton etal., Bioorg Med Chem. 1995 April; 3(4):437-45; Petersen et al., J PeptSci. 1995 May-June; 1(3):175-83; Orum et al., Biotechniques. 1995September; 19(3):472-80; Footer et al., Biochemistry. 1996 Aug. 20;35(33):10673-9; Griffith et al., Nucleic Acids Res. 1995 Aug. 11;23(15):3003-8; Pardridge et al., Proc Natl Acad Sci USA. 1995 Jun. 6;92(12):5592-6; Boffa et al., Proc Natl Acad Sci USA. 1995 Mar. 14;92(6):1901-5; Gambacorti-Passerini et al., Blood. 1996 Aug. 15;88(4):1411-7; Armitage et al., Proc Natl Acad Sci USA. 1997 Nov. 11;94(23):12320-5; Seeger et al., Biotechniques. 1997 September;23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNA chimericmolecules and their uses in diagnostics, modulating protein inorganisms, and treatment of conditions susceptible to therapeutics.

Methods of characterizing the antisense binding properties of PNAs arediscussed in Rose (Anal Chem. 1993 Dec. 15; 65(24):3545-9) and Jensen etal. (Biochemistry. 1997 Apr. 22; 36(16):5072-7). Rose uses capillary gelelectrophoresis to determine binding of PNAs to their complementaryoligonucleotide, measuring the relative binding kinetics andstoichiometry. Similar types of measurements were made by Jensen et al.using BIAcore™ technology.

Other applications of PNAs that have been described and will be apparentto the skilled artisan include use in DNA strand invasion, antisenseinhibition, mutational analysis, enhancers of transcription, nucleicacid purification, isolation of transcriptionally active genes, blockingof transcription factor binding, genome cleavage, biosensors, in situhybridization, and the like.

Polynucleotide Identification, Characterization and Expression

Polynucleotides compositions of the present invention may be identified,prepared and/or manipulated using any of a variety of well establishedtechniques (see generally, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., 1989, and other like references). For example, a polynucleotidemay be identified, as described in more detail below, by screening amicroarray of cDNAs for tumor-associated expression (i.e., expressionthat is at least two fold greater in a tumor than in normal tissue, asdetermined using a representative assay provided herein). Such screensmay be performed, for example, using the microarray technology ofAffymetrix, Inc. (Santa Clara, Calif.) according to the manufacturer'sinstructions (and essentially as described by Schena et al., Proc. Natl.Acad. Sci. USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad.Sci. USA 94:2150-2155, 1997). Alternatively, polynucleotides may beamplified from cDNA prepared from cells expressing the proteinsdescribed herein, such as tumor cells.

Many template dependent processes are available to amplify a targetsequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, each of which is incorporated herein by reference in itsentirety. Briefly, in PCR™, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase (e.g., Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction product and the process isrepeated. Preferably reverse transcription and PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

Any of a number of other template dependent processes, many of which arevariations of the PCR™ amplification technique, are readily known andavailable in the art. Illustratively, some such methods include theligase chain reaction (referred to as LCR), described, for example, inEur. Pat. Appl. Publ. No. 320,308 and U.S. Pat. No. 4,883,750; QbetaReplicase, described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880;Strand Displacement Amplification (SDA) and Repair Chain Reaction (RCR).Still other amplification methods are described in Great Britain Pat.Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No.PCT/US89/01025. Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS) (PCT Intl. Pat. Appl.Publ. No. WO 88/10315), including nucleic acid sequence basedamplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822describes a nucleic acid amplification process involving cyclicallysynthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-strandedDNA (dsDNA). PCT Intl. Pat. Appl. Publ. No. WO 89/06700 describes anucleic acid sequence amplification scheme based on the hybridization ofa promoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Otheramplification methods such as “RACE” (Frohman, 1990), and “one-sidedPCR” (Ohara, 1989) are also well-known to those of skill in the art.

An amplified portion of a polynucleotide of the present invention may beused to isolate a full length gene from a suitable library (e.g., atumor cDNA library) using well known techniques. Within such techniques,a library (cDNA or genomic) is screened using one or more polynucleotideprobes or primers suitable for amplification. Preferably, a library issize-selected to include larger molecules. Random primed libraries mayalso be preferred for identifying 5′ and upstream regions of genes.Genomic libraries are preferred for obtaining introns and extending 5′sequences.

For hybridization techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. cDNA clones may be analyzed to determine the amount ofadditional sequence by, for example, PCR using a primer from the partialsequence and a primer from the vector. Restriction maps and partialsequences may be generated to identify one or more overlapping clones.The complete sequence may then be determined using standard techniques,which may involve generating a series of deletion clones. The resultingoverlapping sequences can then assembled into a single contiguoussequence. A full length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

Alternatively, amplification techniques, such as those described above,can be useful for obtaining a full length coding sequence from a partialcDNA sequence. One such amplification technique is inverse PCR (seeTriglia et al., Nucl. Acids Res. 16:8186, 1988), which uses restrictionenzymes to generate a fragment in the known region of the gene. Thefragment is then circularized by intramolecular ligation and used as atemplate for PCR with divergent primers derived from the known region.Within an alternative approach, sequences adjacent to a partial sequencemay be retrieved by amplification with a primer to a linker sequence anda primer specific to a known region. The amplified sequences aretypically subjected to a second round of amplification with the samelinker primer and a second primer specific to the known region. Avariation on this procedure, which employs two primers that initiateextension in opposite directions from the known sequence, is describedin WO 96/38591. Another such technique is known as “rapid amplificationof cDNA ends” or RACE. This technique involves the use of an internalprimer and an external primer, which hybridizes to a polyA region orvector sequence, to identify sequences that are 5′ and 3′ of a knownsequence. Additional techniques include capture PCR (Lagerstrom et al.,PCR Methods Applic. 1:111-19, 1991) and walking PCR (Parker et al.,Nucl. Acids. Res. 19:3055-60, 1991). Other methods employingamplification may also be employed to obtain a full length cDNAsequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

In other embodiments of the invention, polynucleotide sequences orfragments thereof which encode polypeptides of the invention, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of a polypeptide in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al.(1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the proteinitself may be produced using chemical methods to synthesize the aminoacid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge, J. Y. et al. (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin Elmer, Palo Alto, Calif.).

A newly synthesized peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, T. (1983)Proteins, Structures and Molecular Principles, WH Freeman and Co., NewYork, N.Y.) or other comparable techniques available in the art. Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure).Additionally, the amino acid sequence of a polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

In order to express a desired polypeptide, the nucleotide sequencesencoding the polypeptide, or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described, for example, in Sambrook,J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

A variety of expression vector/host systems may be utilized to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions--whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, any of a number of expression vectors may beselected depending upon the use intended for the expressed polypeptide.For example, when large quantities are needed, for example for theinduction of antibodies, vectors which direct high level expression offusion proteins that are readily purified may be used. Such vectorsinclude, but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of .beta.-galactosidase so that a hybrid proteinis produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhard,E. K. et al. (1994) Proc. Natl. Acad. Sci. 91 :3224-3227).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) geneswhich can be employed in tk.sup.- or aprt.sup.-cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). Theuse of visible markers has gained popularity with such markers asanthocyanins, beta-glucuronidase and its substrate GUS, and luciferaseand its substrate luciferin, being widely used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system (Rhodes, C.A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells that contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include, for example, membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on a given polypeptide may be preferred forsome applications, but a competitive binding assay may also be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.12:441-453).

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield J. (1963) J. Am.Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

Antibody Compositions, Fragments Thereof and Other Binding Agents

According to another aspect, the present invention further providesbinding agents, such as antibodies and antigen-binding fragmentsthereof, that exhibit immunological binding to a tumor polypeptidedisclosed herein, or to a portion, variant or derivative thereof. Anantibody, or antigen-binding fragment thereof, is said to “specificallybind,” “immunogically bind,” and/or is “immunologically reactive” to apolypeptide of the invention if it reacts at a detectable level (within,for example, an ELISA assay) with the polypeptide, and does not reactdetectably with unrelated polypeptides under similar conditions.

Immunological binding, as used in this context, generally refers to thenon-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. The strength, or affinity of immunological bindinginteractions can be expressed in terms of the dissociation constant(K_(d)) of the interaction, wherein a smaller K_(d) represents a greateraffinity. Immunological binding properties of selected polypeptides canbe quantified using methods well known in the art. One such methodentails measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (K_(on)) and the “off rateconstant” (K_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity, and is thus equal to the dissociation constantK_(d). See, generally, Davies et al. (1990) Annual Rev. Biochem.59:439-473.

An “antigen-binding site,” or “binding portion” of an antibody refers tothe part of the immunoglobulin molecule that participates in antigenbinding. The antigen binding site is formed by amino acid residues ofthe N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”)chains. Three highly divergent stretches within the V regions of theheavy and light chains are referred to as “hypervariable regions” whichare interposed between more conserved flanking stretches known as“framework regions,” or “FRs”. Thus the term “FR” refers to amino acidsequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other in threedimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.”

Binding agents may be further capable of differentiating betweenpatients with and without a cancer, such as lung cancer, using therepresentative assays provided herein. For example, antibodies or otherbinding agents that bind to a tumor protein will preferably generate asignal indicating the presence of a cancer in at least about 20% ofpatients with the disease, more preferably at least about 30% ofpatients. Alternatively, or in addition, the antibody will generate anegative signal indicating the absence of the disease in at least about90% of individuals without the cancer. To determine whether a bindingagent satisfies this requirement, biological samples (e.g., blood, sera,sputum, urine and/or tumor biopsies) from patients with and without acancer (as determined using standard clinical tests) may be assayed asdescribed herein for the presence of polypeptides that bind to thebinding agent. Preferably, a statistically significant number of sampleswith and without the disease will be assayed. Each binding agent shouldsatisfy the above criteria; however, those of ordinary skill in the artwill recognize that binding agents may be used in combination to improvesensitivity.

Any agent that satisfies the above requirements may be a binding agent.For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for an antigenic polypeptide of interestmay be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest as compared to one or morecontrol polypeptides). Such cell lines may be produced, for example,from spleen cells obtained from an animal immunized as described above.The spleen cells are then immortalized by, for example, fusion with amyeloma cell fusion partner, preferably one that is syngeneic with theimmunized animal. A variety of fusion techniques may be employed. Forexample, the spleen cells and myeloma cells may be combined with anonionic detergent for a few minutes and then plated at low density on aselective medium that supports the growth of hybrid cells, but notmyeloma cells. A preferred selection technique uses HAT (hypoxanthine,aminopterin, thymidine) selection. After a sufficient time, usuallyabout 1 to 2 weeks, colonies of hybrids are observed. Single coloniesare selected and their culture supernatants tested for binding activityagainst the polypeptide. Hybridomas having high reactivity andspecificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

A number of therapeutically useful molecules are known in the art whichcomprise antigen-binding sites that are capable of exhibitingimmunological binding properties of an antibody molecule. Theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′)₂” fragment which comprises bothantigen-binding sites. An “Fv” fragment can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single chain Fv (“sFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Anumber of methods have been described to discern chemical structures forconverting the naturally aggregated—but chemically separated—light andheavy polypeptide chains from an antibody V region into an sFv moleculewhich will fold into a three dimensional structure substantially similarto the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

Each of the above-described molecules includes a heavy chain and a lightchain CDR set, respectively interposed between a heavy chain and a lightchain FR set which provide support to the CDRS and define the spatialrelationship of the CDRs relative to each other. As used herein, theterm “CDR set” refers to the three hypervariable regions of a heavy orlight chain V region. Proceeding from the N-terminus of a heavy or lightchain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) isreferred to herein as a “molecular recognition unit.” Crystallographicanalysis of a number of antigen-antibody complexes has demonstrated thatthe amino acid residues of CDRs form extensive contact with boundantigen, wherein the most extensive antigen contact is with the heavychain CDR3. Thus, the molecular recognition units are primarilyresponsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRS. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues.

When the V regions fold into a binding-site, the CDRs are displayed asprojecting loop motifs which form an antigen-binding surface. It isgenerally recognized that there are conserved structural regions of FRswhich influence the folded shape of the CDR loops into certain“canonical” structures—regardless of the precise CDR amino acidsequence. Further, certain FR residues are known to participate innon-covalent interdomain contacts which stabilize the interaction of theantibody heavy and light chains.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated CDRs fused to human constant domains (Winter et al. (1991)Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown etal. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a humansupporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients.

As used herein, the terms “veneered FRs” and “recombinantly veneeredFRs” refer to the selective replacement of FR residues from, e.g., arodent heavy or light chain V region, with human FR residues in order toprovide a xenogeneic molecule comprising an antigen-binding site whichretains substantially all of the native FR polypeptide foldingstructure. Veneering techniques are based on the understanding that theligand binding characteristics of an antigen-binding site are determinedprimarily by the structure and relative disposition of the heavy andlight chain CDR sets within the antigen-binding surface. Davies et al.(1990) Ann. Rev. Biochem. 59:439-473. Thus, antigen binding specificitycan be preserved in a humanized antibody only wherein the CDRstructures, their interaction with each other, and their interactionwith the rest of the V region domains are carefully maintained. By usingveneering techniques, exterior (e.g., solvent-accessible) FR residueswhich are readily encountered by the immune system are selectivelyreplaced with human residues to provide a hybrid molecule that compriseseither a weakly immunogenic, or substantially non-immunogenic veneeredsurface.

The process of veneering makes use of the available sequence data forhuman antibody variable domains compiled by Kabat et al., in Sequencesof Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Healthand Human Services, U.S. Government Printing Office, 1987), updates tothe Kabat database, and other accessible U.S. and foreign databases(both nucleic acid and protein).

Solvent accessibilities of V region amino acids can be deduced from theknown three-dimensional structure for human and murine antibodyfragments. There are two general steps in veneering a murineantigen-binding site. Initially, the FRs of the variable domains of anantibody molecule of interest are compared with corresponding FRsequences of human variable domains obtained from the above-identifiedsources. The most homologous human V regions are then compared residueby residue to corresponding murine amino acids. The residues in themurine FR which differ from the human counterpart are replaced by theresidues present in the human moiety using recombinant techniques wellknown in the art. Residue switching is only carried out with moietieswhich are at least partially exposed (solvent accessible), and care isexercised in the replacement of amino acid residues which may have asignificant effect on the tertiary structure of V region domains, suchas proline, glycine and charged amino acids.

In this manner, the resultant “veneered” murine antigen-binding sitesare thus designed to retain the murine CDR residues, the residuessubstantially adjacent to the CDRs, the residues identified as buried ormostly buried (solvent inaccessible), the residues believed toparticipate in non-covalent (e.g., electrostatic and hydrophobic)contacts between heavy and light chain domains, and the residues fromconserved structural regions of the FRs which are believed to influencethe “canonical” tertiary structures of the CDR loops. These designcriteria are then used to prepare recombinant nucleotide sequences whichcombine the CDRs of both the heavy and light chain of a murineantigen-binding site into human-appearing FRs that can be used totransfect mammalian cells for the expression of recombinant humanantibodies which exhibit the antigen specificity of the murine antibodymolecule.

In another embodiment of the invention, monoclonal antibodies of thepresent invention may be coupled to one or more therapeutic agents.Suitable agents in this regard include radionuclides, differentiationinducers, drugs, toxins, and derivatives thereof. Preferredradionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and²¹²Bi. Preferred drugs include methotrexate, and pyrimidine and purineanalogs. Preferred differentiation inducers include phorbol esters andbutyric acid. Preferred toxins include ricin, abrin, diptheria toxin,cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, andpokeweed antiviral protein.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group. Suitable carriers includeproteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato etal.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat.No. 4,699,784, to Shih et al.). A carrier may also bear an agent bynoncovalent bonding or by encapsulation, such as within a liposomevesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriersspecific for radionuclide agents include radiohalogenated smallmolecules and chelating compounds. For example, U.S. Pat. No. 4,735,792discloses representative radiohalogenated small molecules and theirsynthesis. A radionuclide chelate may be formed from chelating compoundsthat include those containing nitrogen and sulfur atoms as the donoratoms for binding the metal, or metal oxide, radionuclide. For example,U.S. Pat. No. 4,673,562, to Davison et al. discloses representativechelating compounds and their synthesis.

T Cell Compositions

The present invention, in another aspect, provides T cells specific fora tumor polypeptide disclosed herein, or for a variant or derivativethereof. Such cells may generally be prepared in vitro or ex vivo, usingstandard procedures. For example, T cells may be isolated from bonemarrow, peripheral blood, or a fraction of bone marrow or peripheralblood of a patient, using a commercially available cell separationsystem, such as the Isolex™ System, available from Nexell Therapeutics,Inc. (Irvine, Calif.; see also U.S. Pat. No. 5,240,856; U.S. Pat. No.5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, Tcells may be derived from related or unrelated humans, non-humanmammals, cell lines or cultures.

T cells may be stimulated with a polypeptide, polynucleotide encoding apolypeptide and/or an antigen presenting cell (APC) that expresses sucha polypeptide. Such stimulation is performed under conditions and for atime sufficient to permit the generation of T cells that are specificfor the polypeptide of interest. Preferably, a tumor polypeptide orpolynucleotide of the invention is present within a delivery vehicle,such as a microsphere, to facilitate the generation of specific T cells.

T cells are considered to be specific for a polypeptide of the presentinvention if the T cells specifically proliferate, secrete cytokines orkill target cells coated with the polypeptide or expressing a geneencoding the polypeptide. T cell specificity may be evaluated using anyof a variety of standard techniques. For example, within a chromiumrelease assay or proliferation assay, a stimulation index of more thantwo fold increase in lysis and/or proliferation, compared to negativecontrols, indicates T cell specificity. Such assays may be performed,for example, as described in Chen et al., Cancer Res. 54:1065-1070,1994. Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labeling cultures of T cells with tritiatedthymidine and measuring the amount of tritiated thymidine incorporatedinto DNA). Contact with a tumor polypeptide (100 ng/ml-100 μg/ml,preferably 200 ng/ml-25 μg/ml) for 3-7 days will typically result in atleast a two fold increase in proliferation of the T cells. Contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells thathave been activated in response to a tumor polypeptide, polynucleotideor polypeptide-expressing APC may be CD4⁺ and/or CD8⁺. Tumorpolypeptide-specific T cells may be expanded using standard techniques.Within preferred embodiments, the T cells are derived from a patient, arelated donor or an unrelated donor, and are administered to the patientfollowing stimulation and expansion.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to a tumor polypeptide, polynucleotide or APC can be expandedin number either in vitro or in vivo. Proliferation of such T cells invitro may be accomplished in a variety of ways. For example, the T cellscan be re-exposed to a tumor polypeptide, or a short peptidecorresponding to an immunogenic portion of such a polypeptide, with orwithout the addition of T cell growth factors, such as interleukin-2,and/or stimulator cells that synthesize a tumor polypeptide.Alternatively, one or more T cells that proliferate in the presence ofthe tumor polypeptide can be expanded in number by cloning. Methods forcloning cells are well known in the art, and include limiting dilution.

T Cell Receptor Compositions

The T cell receptor (TCR) consists of 2 different, highly variablepolypeptide chains, termed the T-cell receptor α and β chains, that arelinked by a disulfide bond (Janeway, Travers, Walport. Immunobiology.Fourth Ed., 148-159. Elsevier Science Ltd/Garland Publishing. 1999). Theα/β heterodimer complexes with the invariant CD3 chains at the cellmembrane. This complex recognizes specific antigenic peptides bound toMHC molecules. The enormous diversity of TCR specificities is generatedmuch like immunoglobulin diversity, through somatic gene rearrangement.The β chain genes contain over 50 variable (V), 2 diversity (D), over 10joining (J) segments, and 2 constant region segments (C). The α chaingenes contain over 70 V segments, and over 60 J segments but no Dsegments, as well as one C segment. During T cell development in thethymus, the D to J gene rearrangement of the β chain occurs, followed bythe V gene segment rearrangement to the DJ. This functional VDJ_(β) exonis transcribed and spliced to join to a C_(β). For the a chain, a V_(α)gene segment rearranges to a J_(α) gene segment to create the functionalexon that is then transcribed and spliced to the C_(α). Diversity isfurther increased during the recombination process by the randomaddition of P and N-nucleotides between the V, D, and J segments of theβ chain and between the V and J segments in the a chain (Janeway,Travers, Walport. Immunobiology. Fourth Ed., 98 and 150. ElsevierScience Ltd/Garland Publishing. 1999).

The present invention, in another aspect, provides TCRs specific for apolypeptide disclosed herein, or for a variant or derivative thereof. Inaccordance with the present invention, polynucleotide and amino acidsequences are provided for the V-J or V-D-J junctional regions or partsthereof for the alpha and beta chains of the T-cell receptor whichrecognize tumor polypeptides described herein. In general, this aspectof the invention relates to T-cell receptors which recognize or bindtumor polypeptides presented in the context of MHC. In a preferredembodiment the tumor antigens recognized by the T-cell receptorscomprise a polypeptide of the present invention. For example, cDNAencoding a TCR specific for a_tumor peptide can be isolated from T cellsspecific for a tumor polypeptide using standard molecular biological andrecombinant DNA techniques.

This invention further includes the T-cell receptors or analogs thereofhaving substantially the same function or activity as the T-cellreceptors of this invention which recognize or bind tumor polypeptides.Such receptors include, but are not limited to, a fragment of thereceptor, or a substitution, addition or deletion mutant of a T-cellreceptor provided herein. This invention also encompasses polypeptidesor peptides that are substantially homologous to the T-cell receptorsprovided herein or that retain substantially the same activity. The term“analog” includes any protein or polypeptide having an amino acidresidue sequence substantially identical to the T-cell receptorsprovided herein in which one or more residues, preferably no more than 5residues, more preferably no more than 25 residues have beenconservatively substituted with a functionally similar residue and whichdisplays the functional aspects of the T-cell receptor as describedherein.

The present invention further provides for suitable mammalian hostcells, for example, non-specific T cells, that are transfected with apolynucleotide encoding TCRs specific for a polypeptide describedherein, thereby rendering the host cell specific for the polypeptide.The α and β chains of the TCR may be contained on separate expressionvectors or alternatively, on a single expression vector that alsocontains an internal ribosome entry site (IRES) for cap-independenttranslation of the gene downstream of the IRES. Said host cellsexpressing TCRs specific for the polypeptide may be used, for example,for adoptive immunotherapy of lung cancer as discussed further below.

In further aspects of the present invention, cloned TCRs specific for apolypeptide recited herein may be used in a kit for the diagnosis oflung cancer. For example, the nucleic acid sequence or portions thereof,of tumor-specific TCRs can be used as probes or primers for thedetection of expression of the rearranged genes encoding the specificTCR in a biological sample. Therefore, the present invention furtherprovides for an assay for detecting messenger RNA or DNA encoding theTCR specific for a polypeptide.

Pharmaceutical Compositions

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide, T-cell and/or antibodycompositions disclosed herein in pharmaceutically-acceptable carriersfor administration to a cell or an animal, either alone, or incombination with one or more other modalities of therapy.

It will be understood that, if desired, a composition as disclosedherein may be administered in combination with other agents as well,such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

Therefore, in another aspect of the present invention, pharmaceuticalcompositions are provided comprising one or more of the polynucleotide,polypeptide, antibody, and/or T-cell compositions described herein incombination with a physiologically acceptable carrier. In certainpreferred embodiments, the pharmaceutical compositions of the inventioncomprise immunogenic polynucleotide and/or polypeptide compositions ofthe invention for use in prophylactic and theraputic vaccineapplications. Vaccine preparation is generally described in, forexample, M. F. Powell and M. J. Newman, eds., “Vaccine Design (thesubunit and adjuvant approach),” Plenum Press (NY, 1995). Generally,such compositions will comprise one or more polynucleotide and/orpolypeptide compositions of the present invention in combination withone or more immunostimulants.

It will be apparent that any of the pharmaceutical compositionsdescribed herein can contain pharmaceutically acceptable salts of thepolynucleotides and polypeptides of the invention. Such salts can beprepared, for example, from pharmaceutically acceptable non-toxic bases,including organic bases (e.g., salts of primary, secondary and tertiaryamines and basic amino acids) and inorganic bases (e.g., sodium,potassium, lithium, ammonium, calcium and magnesium salts).

In another embodiment, illustrative immunogenic compositions, e.g.,vaccine compositions, of the present invention comprise DNA encoding oneor more of the polypeptides as described above, such that thepolypeptide is generated in situ. As noted above, the polynucleotide maybe administered within any of a variety of delivery systems known tothose of ordinary skill in the art. Indeed, numerous gene deliverytechniques are well known in the art, such as those described byRolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, andreferences cited therein. Appropriate polynucleotide expression systemswill, of course, contain the necessary regulatory DNA regulatorysequences for expression in a patient (such as a suitable promoter andterminating signal). Alternatively, bacterial delivery systems mayinvolve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface or secretes such an epitope.

Therefore, in certain embodiments, polynucleotides encoding immunogenicpolypeptides described herein are introduced into suitable mammalianhost cells for expression using any of a number of known viral-basedsystems. In one illustrative embodiment, retroviruses provide aconvenient and effective platform for gene delivery systems. A selectednucleotide sequence encoding a polypeptide of the present invention canbe inserted into a vector and packaged in retroviral particles usingtechniques known in the art. The recombinant virus can then be isolatedand delivered to a subject. A number of illustrative retroviral systemshave been described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman(1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993)Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin(1993) Cur. Opin. Genet. Develop. 3:102-109.

In addition, a number of illustrative adenovirus-based systems have alsobeen described. Unlike retroviruses which integrate into the hostgenome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476). Various adeno-associated virus (AAV)vector systems have also been developed for polynucleotide delivery. AAVvectors can be readily constructed using techniques well known in theart. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; InternationalPublication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al. (1988)Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (ColdSpring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion inBiotechnology 3:533-539; Muzyczka, N. (1992) Current Topics inMicrobiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human GeneTherapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; andZhou et al. (1994) J. Exp. Med. 179:1867-1875.

Additional viral vectors useful for delivering the polynucleotidesencoding polypeptides of the present invention by gene transfer includethose derived from the pox family of viruses, such as vaccinia virus andavian poxvirus. By way of example, vaccinia virus recombinantsexpressing the novel molecules can be constructed as follows. The DNAencoding a polypeptide is first inserted into an appropriate vector sothat it is adjacent to a vaccinia promoter and flanking vaccinia DNAsequences, such as the sequence encoding thymidine kinase (TK). Thisvector is then used to transfect cells which are simultaneously infectedwith vaccinia. Homologous recombination serves to insert the vacciniapromoter plus the gene encoding the polypeptide of interest into theviral genome. The resulting TK.sup.(−) recombinant can be selected byculturing the cells in the presence of 5-bromodeoxyuridine and pickingviral plaques resistant thereto.

A vaccinia-based infection/transfection system can be conveniently usedto provide for inducible, transient expression or coexpression of one ormore polypeptides described herein in host cells of an organism. In thisparticular system, cells are first infected in vitro with a vacciniavirus recombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the polynucleotide or polynucleotides of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAwhich is then translated into polypeptide by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990)87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986)83:8122-8126.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the coding sequences of interest.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a number of alphavirus vectors can also be used for delivery ofpolynucleotide compositions of the present invention, such as thosevectors described in U.S. Pat. Nos. 5,843,723; 6,015,686; 6,008,035 and6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE)can also be used, illustrative examples of which can be found in U.S.Pat. Nos. 5,505,947 and 5,643,576.

Moreover, molecular conjugate vectors, such as the adenovirus chimericvectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, canalso be used for gene delivery under the invention.

Additional illustrative information on these and other known viral-baseddelivery systems can be found, for example, in Fisher-Hoch et al., Proc.Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad.Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat.Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993.

In certain embodiments, a polynucleotide may be integrated into thegenome of a target cell. This integration may be in the specificlocation and orientation via homologous recombination (gene replacement)or it may be integrated in a random, non-specific location (geneaugmentation). In yet further embodiments, the polynucleotide may bestably maintained in the cell as a separate, episomal segment of DNA.Such polynucleotide segments or “episomes” encode sequences sufficientto permit maintenance and replication independent of or insynchronization with the host cell cycle. The manner in which theexpression construct is delivered to a cell and where in the cell thepolynucleotide remains is dependent on the type of expression constructemployed.

In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

In still another embodiment, a composition of the present invention canbe delivered via a particle bombardment approach, many of which havebeen described. In one illustrative example, gas-driven particleacceleration can be achieved with devices such as those manufactured byPowderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc.(Madison, Wis.), some examples of which are described in U.S. Pat. Nos.5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799.This approach offers a needle-free delivery approach wherein a drypowder formulation of microscopic particles, such as polynucleotide orpolypeptide particles, are accelerated to high speed within a helium gasjet generated by a hand held device, propelling the particles into atarget tissue of interest.

In a related embodiment, other devices and methods that may be usefulfor gas-driven needle-less injection of compositions of the presentinvention include those provided by Bioject, Inc. (Portland, Oreg.),some examples of which are described in U.S. Pat. Nos. 4,790,824;5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

According to another embodiment, the pharmaceutical compositionsdescribed herein will comprise one or more immunostimulants in additionto the immunogenic polynucleotide, polypeptide, antibody, T-cell and/orAPC compositions of this invention. An immunostimulant refers toessentially any substance that enhances or potentiates an immuneresponse (antibody and/or cell-mediated) to an exogenous antigen. Onepreferred type of immunostimulant comprises an adjuvant. Many adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Certain adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham,Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A, QS21, aminoalkyl glucosaminide4-phosphates, and quil A. Cytokines, such as GM-CSF, interleukin-2, -7,-12, and other like growth factors, may also be used as adjuvants.

Within certain embodiments of the invention, the adjuvant composition ispreferably one that induces an immune response predominantly of the Th1type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

Certain preferred adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A, together with analuminum salt. MPL® adjuvants are available from Corixa Corporation(Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins .Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol® toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose. In one preferred embodiment, theadjuvant system includes the combination of a monophosphoryl lipid A anda saponin derivative, such as the combination of QS21 and 3D-MPL®adjuvant, as described in WO 94/00153, or a less reactogenic compositionwhere the QS21 is quenched with cholesterol, as described in WO96/33739. Other preferred formulations comprise an oil-in-water emulsionand tocopherol. Another particularly preferred adjuvant formulationemploying QS21, 3D-MPL® adjuvant and tocopherol in an oil-in-wateremulsion is described in WO 95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 is disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

Additional illustrative adjuvants for use in the pharmaceuticalcompositions of the invention include Montanide ISA 720 (Seppic,France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59(Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium), Detox(Enhanzyn®) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720, the disclosures of which are incorporated herein byreference in their entireties, and polyoxyethylene ether adjuvants suchas those described in WO 99/52549A1.

Other preferred adjuvants include adjuvant molecules of the generalformula

HO(CH₂CH₂O)_(n)-A-R,   (I):

wherein, n is 1-50, A is a bond or —C(O)—, R is C₁-₅₀ alkyl or PhenylC₁₋₅₀ alkyl.

One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

The polyoxyethylene ether according to the general formula (I) abovemay, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

According to another embodiment of this invention, an immunogeniccomposition described herein is delivered to a host via antigenpresenting cells (APCs), such as dendritic cells, macrophages, B cells,monocytes and other cells that may be engineered to be efficient APCs.Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-tumor effects per seand/or to be immunologically compatible with the receiver (i.e., matchedHLA haplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatena{dot over (i)}ve T cell responses. Dendritic cells may, of course, beengineered to express specific cell-surface receptors or ligands thatare not commonly found on dendritic cells in vivo or ex vivo, and suchmodified dendritic cells are contemplated by the present invention. Asan alternative to dendritic cells, secreted vesicles antigen-loadeddendritic cells (called exosomes) may be used within a vaccine (seeZitvogel et al., Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide of the invention(or portion or other variant thereof) such that the encoded polypeptide,or an immunogenic portion thereof, is expressed on the cell surface.Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the tumor polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will typically vary depending on the mode ofadministration. Compositions of the present invention may be formulatedfor any appropriate manner of administration, including for example,topical, oral, nasal, mucosal, intravenous, intracranial,intraperitoneal, subcutaneous and intramuscular administration.

Carriers for use within such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation preferably provides a relatively constant level ofactive component release. In other embodiments, however, a more rapidrate of release immediately upon administration may be desired. Theformulation of such compositions is well within the level of ordinaryskill in the art using known techniques. Illustrative carriers useful inthis regard include microparticles of poly(lactide-co-glycolide),polyacrylate, latex, starch, cellulose, dextran and the like. Otherillustrative delayed-release carriers include supramolecular biovectors,which comprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g.,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

In another illustrative embodiment, biodegradable microspheres (e.g.,polylactate polyglycolate) are employed as carriers for the compositionsof this invention. Suitable biodegradable microspheres are disclosed,for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647;5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252.Modified hepatitis B core protein carrier systems. such as described inWO/99 40934, and references cited therein, will also be useful for manyapplications. Another illustrative carrier/delivery system employs acarrier comprising particulate-protein complexes, such as thosedescribed in U.S. Pat. No. 5,928,647, which are capable of inducing aclass I-restricted cytotoxic T lymphocyte responses in a host.

The pharmaceutical compositions of the invention will often furthercomprise one or more buffers (e.g., neutral buffered saline or phosphatebuffered saline), carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation, is well known in the art,some of which are briefly discussed below for general purposes ofillustration.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (see, for example,Mathiowitz et al., Nature 1997 Mar. 27; 386(6623):410-4; Hwang et al.,Crit Rev Ther Drug Carrier Syst 1998; 15(3):243-84; U.S. Pat. No.5,641,515; U. S. Patent 5,580,579 and U.S. Pat. No. 5,792,451). Tablets,troches, pills, capsules and the like may also contain any of a varietyof additional components, for example, a binder, such as gum tragacanth,acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate;a disintegrating agent, such as corn starch, potato starch, alginic acidand the like; a lubricant, such as magnesium stearate; and a sweeteningagent, such as sucrose, lactose or saccharin may be added or a flavoringagent, such as peppermint, oil of wintergreen, or cherry flavoring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. Of course, any material used in preparingany dosage unit form should be pharmaceutically pure and substantiallynon-toxic in the amounts employed. In addition, the active compounds maybe incorporated into sustained-release preparation and formulations.

Typically, these formulations will contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. Alternatively, the active ingredientmay be incorporated into an oral solution such as one containing sodiumborate, glycerin and potassium bicarbonate, or dispersed in adentifrice, or added in a therapeutically-effective amount to acomposition that may include water, binders, abrasives, flavoringagents, foaming agents, and humectants. Alternatively the compositionsmay be fashioned into a tablet or solution form that may be placed underthe tongue or otherwise dissolved in the mouth.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat.No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. Moreover, for human administration, preparationswill of course preferably meet sterility, pyrogenicity, and the generalsafety and purity standards as required by FDA Office of Biologicsstandards.

In another embodiment of the invention, the compositions disclosedherein may be formulated in a neutral or salt form. Illustrativepharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212.Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., J Controlled Release 1998 Mar. 2; 52(1-2):81-7) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are alsowell-known in the pharmaceutical arts. Likewise, illustrativetransmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol 1998 July; 16(7):307-21;Takakura, Nippon Rinsho 1998 March; 56(3):691-5; Chandran et al., IndianJ Exp Biol. 1997 August; 35(8):801-9; Margalit, Crit Rev Ther DrugCarrier Syst. 1995; 12(2-3):233-61; U.S. Pat. No. 5,567,434; U.S. Pat.No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety).

Liposomes have been used successfully with a number of cell types thatare normally difficult to transfect by other procedures, including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J Biol Chem. 1990 Sep. 25; 265(27):16337-42; Muller et al., DNACell Biol. 1990 April; 9(3):221-9). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes,various drugs, radiotherapeutic agents, enzymes, viruses, transcriptionfactors, allosteric effectors and the like, into a variety of culturedcell lines and animals. Furthermore, he use of liposomes does not appearto be associated with autoimmune responses or unacceptable toxicityafter systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that aredispersed in an aqueous medium and spontaneously form multilamellarconcentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. 1998 December; 24(12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 March;45(2):149-55; Zambaux et al. J Controlled Release. 1998 Jan. 2;50(1-3):31-40; and U.S. Pat. No. 5,145,684.

Cancer Therapeutic Methods

Immunologic approaches to cancer therapy are based on the recognitionthat cancer cells can often evade the body's defenses against aberrantor foreign cells and molecules, and that these defenses might betherapeutically stimulated to regain the lost ground, e.g. pgs. 623-648in Klein, Immunology (Wiley-Interscience, New York, 1982). Numerousrecent observations that various immune effectors can directly orindirectly inhibit growth of tumors has led to renewed interest in thisapproach to cancer therapy, e.g. Jager, et al., Oncology 2001;60(1):1-7; Renner, et al., Ann Hematol 2000 December; 79(12):651-9.

Four-basic cell types whose function has been associated with antitumorcell immunity and the elimination of tumor cells from the body are: i)B-lymphocytes which secrete immunoglobulins into the blood plasma foridentifying and labeling the nonself invader cells; ii) monocytes whichsecrete the complement proteins that are responsible for lysing andprocessing the immunoglobulin-coated target invader cells; iii) naturalkiller lymphocytes having two mechanisms for the destruction of tumorcells, antibody-dependent cellular cytotoxicity and natural killing; andiv) T-lymphocytes possessing antigen-specific receptors and having thecapacity to recognize a tumor cell carrying complementary markermolecules (Schreiber, H., 1989, in Fundamental Immunology (ed). W. E.Paul, pp. 923-955).

Cancer immunotherapy generally focuses on inducing humoral immuneresponses, cellular immune responses, or both. Moreover, it is wellestablished that induction of CD4⁺ T helper cells is necessary in orderto secondarily induce either antibodies or cytotoxic CD8⁺ T cells.Polypeptide antigens that are selective or ideally specific for cancercells, particularly lung cancer cells, offer a powerful approach forinducing immune responses against lung cancer, and are an importantaspect of the present invention.

Therefore, in further aspects of the present invention, thepharmaceutical compositions described herein may be used for thetreatment of cancer, particularly for the immunotherapy of lung cancer.Within such methods, the pharmaceutical compositions described hereinare administered to a patient, typically a warm-blooded animal,preferably a human. A patient may or may not be afflicted with cancer.Accordingly, the above pharmaceutical compositions may be used toprevent the development of a cancer or to treat a patient afflicted witha cancer. Pharmaceutical compositions and vaccines may be administeredeither prior to or following surgical removal of primary tumors and/ortreatment such as administration of radiotherapy or conventionalchemotherapeutic drugs. As discussed above, administration of thepharmaceutical compositions may be by any suitable method, includingadministration by intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal, intradermal, anal, vaginal, topical and oralroutes.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against tumors with the administration ofimmune response-modifying agents (such as polypeptides andpolynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as effector cells or antibodies) that candirectly or indirectly mediate antitumor effects and does notnecessarily depend on an intact host immune system. Examples of effectorcells include T cells as discussed above, T lymphocytes (such as CD8⁺cytotoxic T lymphocytes and CD4⁺ T-helper tumor-infiltratinglymphocytes), killer cells (such as Natural Killer cells andlymphokine-activated killer cells), B cells and antigen-presenting cells(such as dendritic cells and macrophages) expressing a polypeptideprovided herein. T cell receptors and antibody receptors specific forthe polypeptides recited herein may be cloned, expressed and transferredinto other vectors or effector cells for adoptive immunotherapy. Thepolypeptides provided herein may also be used to generate antibodies oranti-idiotypic antibodies (as described above and in U.S. Pat. No.4,918,164) for passive immunotherapy.

Monoclonal antibodies may be labeled with any of a variety of labels fordesired selective usages in detection, diagnostic assays or therapeuticapplications (as described in U.S. Pat. Nos. 6,090,365; 6,015,542;5,843,398; 5,595,721; and 4,708,930, hereby incorporated by reference intheir entirety as if each was incorporated individually). In each case,the binding of the labelled monoclonal antibody to the determinant siteof the antigen will signal detection or delivery of a particulartherapeutic agent to the antigenic determinant on the non-normal cell. Afurther object of this invention is to provide the specific monoclonalantibody suitably labelled for achieving such desired selective usagesthereof.

Effector cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

Alternatively, a vector expressing a polypeptide recited herein may beintroduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal or intratumor administration. Routes andfrequency of administration of the therapeutic compositions describedherein, as well as dosage, will vary from individual to individual, andmay be readily established using standard techniques. In general, thepharmaceutical compositions and vaccines may be administered byinjection (e.g., intracutaneous, intramuscular, intravenous orsubcutaneous), intranasally (e.g., by aspiration) or orally. Preferably,between 1 and 10 doses may be administered over a 52 week period.Preferably, 6 doses are administered, at intervals of 1 month, andbooster vaccinations may be given periodically thereafter. Alternateprotocols may be appropriate for individual patients. A suitable dose isan amount of a compound that, when administered as described above, iscapable of promoting an anti-tumor immune response, and is at least10-50% above the basal (i.e., untreated) level. Such response can bemonitored by measuring the anti-tumor antibodies in a patient or byvaccine-dependent generation of cytolytic effector cells capable ofkilling the patient's tumor cells in vitro. Such vaccines should also becapable of causing an immune response that leads to an improved clinicaloutcome (e.g., more frequent remissions, complete or partial or longerdisease-free survival) in vaccinated patients as compared tonon-vaccinated patients. In general, for pharmaceutical compositions andvaccines comprising one or more polypeptides, the amount of eachpolypeptide present in a dose ranges from about 25 μg to 5 mg per kg ofhost. Suitable dose sizes will vary with the size of the patient, butwill typically range from about 0.1 mL to about 5 mL.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated patients ascompared to non-treated patients. Increases in preexisting immuneresponses to a tumor protein generally correlate with an improvedclinical outcome. Such immune responses may generally be evaluated usingstandard proliferation, cytotoxicity or cytokine assays, which may beperformed using samples obtained from a patient before and aftertreatment.

Cancer Detection and Diagnostic Compositions, Methods and Kits

In general, a cancer may be detected in a patient based on the presenceof one or more lung tumor proteins and/or polynucleotides encoding suchproteins in a biological sample (for example, blood, sera, sputum urineand/or tumor biopsies) obtained from the patient. In other words, suchproteins may be used as markers to indicate the presence or absence of acancer such as lung cancer. In addition, such proteins may be useful forthe detection of other cancers. The binding agents provided hereingenerally permit detection of the level of antigen that binds to theagent in the biological sample.

Polynucleotide primers and probes may be used to detect the level ofmRNA encoding a tumor protein, which is also indicative of the presenceor absence of a cancer. In general, a tumor sequence should be presentat a level that is at least two-fold, preferably three-fold, and morepreferably five-fold or higher in tumor tissue than in normal tissue ofthe same type from which the tumor arose. Expression levels of aparticular tumor sequence in tissue types different from that in whichthe tumor arose are irrelevant in certain diagnostic embodiments sincethe presence of tumor cells can be confirmed by observation ofpredetermined differential expression levels, e.g., 2-fold, 5-fold, etc,in tumor tissue to expression levels in normal tissue of the same type.

Other differential expression patterns can be utilized advantageouslyfor diagnostic purposes. For example, in one aspect of the invention,overexpression of a tumor sequence in tumor tissue and normal tissue ofthe same type, but not in other normal tissue types, e.g. PBMCs, can beexploited diagnostically. In this case, the presence of metastatic tumorcells, for example in a sample taken from the circulation or some othertissue site different from that in which the tumor arose, can beidentified and/or confirmed by detecting expression of the tumorsequence in the sample, for example using RT-PCR analysis. In manyinstances, it will be desired to enrich for tumor cells in the sample ofinterest, e.g., PBMCs, using cell capture or other like techniques.

There are a variety of assay formats known to those of ordinary skill inthe art for using a binding agent to detect polypeptide markers in asample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor

Laboratory, 1988. In general, the presence or absence of a cancer in apatient may be determined by (a) contacting a biological sample obtainedfrom a patient with a binding agent; (b) detecting in the sample a levelof polypeptide that binds to the binding agent; and (c) comparing thelevel of polypeptide with a predetermined cut-off value.

In a preferred embodiment, the assay involves the use of binding agentimmobilized on a solid support to bind to and remove the polypeptidefrom the remainder of the sample. The bound polypeptide may then bedetected using a detection reagent that contains a reporter group andspecifically binds to the binding agent/polypeptide complex. Suchdetection reagents may comprise, for example, a binding agent thatspecifically binds to the polypeptide or an antibody or other agent thatspecifically binds to the binding agent, such as an anti-immunoglobulin,protein G, protein A or a lectin. Alternatively, a competitive assay maybe utilized, in which a polypeptide is labeled with a reporter group andallowed to bind to the immobilized binding agent after incubation of thebinding agent with the sample. The extent to which components of thesample inhibit the binding of the labeled polypeptide to the bindingagent is indicative of the reactivity of the sample with the immobilizedbinding agent. Suitable polypeptides for use within such assays includefull length lung tumor proteins and polypeptide portions thereof towhich the binding agent binds, as described above.

The solid support may be any material known to those of ordinary skillin the art to which the tumor protein may be attached. For example, thesolid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the binding agent. For example, the bindingagent may be covalently attached to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on the bindingpartner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991,at A12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. Thisassay may be performed by first contacting an antibody that has beenimmobilized on a solid support, commonly the well of a microtiter plate,with the sample, such that polypeptides within the sample are allowed tobind to the immobilized antibody. Unbound sample is then removed fromthe immobilized polypeptide-antibody complexes and a detection reagent(preferably a second antibody capable of binding to a different site onthe polypeptide) containing a reporter group is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or Tween 20™(Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is thenincubated with the sample, and polypeptide is allowed to bind to theantibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of polypeptide within a sampleobtained from an individual with lung cancer. Preferably, the contacttime is sufficient to achieve a level of binding that is at least about95% of that achieved at equilibrium between bound and unboundpolypeptide. Those of ordinary skill in the art will recognize that thetime necessary to achieve equilibrium may be readily determined byassaying the level of binding that occurs over a period of time. At roomtemperature, an incubation time of about 30 minutes is generallysufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™. The secondantibody, which contains a reporter group, may then be added to thesolid support. Preferred reporter groups include those groups recitedabove.

The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of a cancer, such as lung cancer,the signal detected from the reporter group that remains bound to thesolid support is generally compared to a signal that corresponds to apredetermined cut-off value. In one preferred embodiment, the cut-offvalue for the detection of a cancer is the average mean signal obtainedwhen the immobilized antibody is incubated with samples from patientswithout the cancer. In general, a sample generating a signal that isthree standard deviations above the predetermined cut-off value isconsidered positive for the cancer. In an alternate preferredembodiment, the cut-off value is determined using a Receiver OperatorCurve, according to the method of Sackett et al., Clinical Epidemiology:A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p.106-7. Briefly, in this embodiment, the cut-off value may be determinedfrom a plot of pairs of true positive rates (i.e., sensitivity) andfalse positive rates (100%-specificity) that correspond to each possiblecut-off value for the diagnostic test result. The cut-off value on theplot that is the closest to the upper left-hand corner (i.e., the valuethat encloses the largest area) is the most accurate cut-off value, anda sample generating a signal that is higher than the cut-off valuedetermined by this method may be considered positive. Alternatively, thecut-off value may be shifted to the left along the plot, to minimize thefalse positive rate, or to the right, to minimize the false negativerate. In general, a sample generating a signal that is higher than thecut-off value determined by this method is considered positive for acancer.

In a related embodiment, the assay is performed in a flow-through orstrip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of a cancer. Typically, the concentration of second bindingagent at that site generates a pattern, such as a line, that can be readvisually. The absence of such a pattern indicates a negative result. Ingeneral, the amount of binding agent immobilized on the membrane isselected to generate a visually discernible pattern when the biologicalsample contains a level of polypeptide that would be sufficient togenerate a positive signal in the two-antibody sandwich assay, in theformat discussed above. Preferred binding agents for use in such assaysare antibodies and antigen-binding fragments thereof. Preferably, theamount of antibody immobilized on the membrane ranges from about 25 ngto about 14, and more preferably from about 50 ng to about 500 ng. Suchtests can typically be performed with a very small amount of biologicalsample.

Of course, numerous other assay protocols exist that are suitable foruse with the tumor proteins or binding agents of the present invention.The above descriptions are intended to be exemplary only. For example,it will be apparent to those of ordinary skill in the art that the aboveprotocols may be readily modified to use tumor polypeptides to detectantibodies that bind to such polypeptides in a biological sample. Thedetection of such tumor protein specific antibodies may correlate withthe presence of a cancer.

A cancer may also, or alternatively, be detected based on the presenceof T cells that specifically react with a tumor protein in a biologicalsample. Within certain methods, a biological sample comprising CD4⁺and/or CD8⁺ T cells isolated from a patient is incubated with a tumorpolypeptide, a polynucleotide encoding such a polypeptide and/or an APCthat expresses at least an immunogenic portion of such a polypeptide,and the presence or absence of specific activation of the T cells isdetected. Suitable biological samples include, but are not limited to,isolated T cells. For example, T cells may be isolated from a patient byroutine techniques (such as by Ficoll/Hypaque density gradientcentrifugation of peripheral blood lymphocytes). T cells may beincubated in vitro for 2-9 days (typically 4 days) at 37° C. withpolypeptide (e.g., 5 -25 μg/ml). It may be desirable to incubate anotheraliquot of a T cell sample in the absence of tumor polypeptide to serveas a control. For CD4⁺ T cells, activation is preferably detected byevaluating proliferation of the T cells. For CD8⁺ T cells, activation ispreferably detected by evaluating cytolytic activity. A level ofproliferation that is at least two fold greater and/or a level ofcytolytic activity that is at least 20% greater than in disease-freepatients indicates the presence of a cancer in the patient.

As noted above, a cancer may also, or alternatively, be detected basedon the level of mRNA encoding a tumor protein in a biological sample.For example, at least two oligonucleotide primers may be employed in apolymerase chain reaction (PCR) based assay to amplify a portion of atumor cDNA derived from a biological sample, wherein at least one of theoligonucleotide primers is specific for a polynucleotide encoding thetumor protein. The amplified cDNA is then separated and detected usingtechniques well known in the art, such as gel electrophoresis.

As would be readily understood by the skilled artisan, “specific for” isa term of art. Determining whether and the conditions under which anoligonucleotide primer or probe is specific for a particular sequence ofinterest can easily be determined by routine experimentation using anynumber of assays known in the art, such as PCR, RT-PCR, andhybridization assays such as Northern Blots. That is, any number ofassays can be used to determine the appropriate conditions and theappropriate oligonucleotide sequence such that the primer amplifies orthe probe hybridizes to a sequence of interest but does not amplify orhybridize to one or more irrelevant control sequences. Further, avariety of computer programs are available in the art that can be usedto design specific primers and probes. It should be noted that theolignucleotide primers and probes need not be 100% identical to thetarget sequence of interest in order to be specific for that sequence.As would be understood by the skilled artisan, mismatches are tolerated.Oligonucleotide primers and probes can have 99%, 98%, 97%, 96%, 95%,94%, 93%, 92%, 91%, 90%, and lower identity to the target sequence ofinterest. Furthermore, as would be understood by the skilled artisan,primers and probes can include additional sequence, such as restrictionendonuclease cleavage sites, that is not complementary to the sequenceof interest, without impacting specificity of the primer or probe.

Similarly, oligonucleotide probes that specifically hybridize to apolynucleotide encoding a tumor protein may be used in a hybridizationassay to detect the presence of polynucleotide encoding the tumorprotein in a biological sample.

To permit hybridization under assay conditions, oligonucleotide primersand probes should comprise an oligonucleotide sequence that has at leastabout 60%, preferably at least about 75% and more preferably at leastabout 90%, identity to a portion of a polynucleotide encoding a tumorprotein of the invention that is at least 10 nucleotides, and preferablyat least 20 nucleotides, in length. Preferably, oligonucleotide primersand/or probes hybridize to a polynucleotide encoding a polypeptidedescribed herein under moderately stringent conditions, as definedabove. Oligonucleotide primers and/or probes which may be usefullyemployed in the diagnostic methods described herein preferably are atleast 10-40 nucleotides in length. In a preferred embodiment, theoligonucleotide primers comprise at least 10 contiguous nucleotides,more preferably at least 15 contiguous nucleotides, of a DNA moleculehaving a sequence as disclosed herein. Techniques for both PCR basedassays and hybridization assays are well known in the art (see, forexample, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263,1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989).

One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample, such as biopsy tissue, and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not afflicted with a cancer. The amplification reaction may beperformed on several dilutions of cDNA spanning two orders of magnitude.A two-fold or greater increase in expression in several dilutions of thetest patient sample as compared to the same dilutions of thenon-cancerous sample is typically considered positive.

In another aspect of the present invention, cell capture technologiesmay be used in conjunction, with, for example, real-time PCR to providea more sensitive tool for detection of metastatic cells expressing lungtumor antigens. Detection of lung cancer cells in biological samples,e.g., bone marrow samples, peripheral blood, and small needle aspirationsamples is desirable for diagnosis and prognosis in lung cancerpatients.

Immunomagnetic beads coated with specific monoclonal antibodies tosurface cell markers, or tetrameric antibody complexes, may be used tofirst enrich or positively select cancer cells in a sample. Variouscommercially available kits may be used, including Dynabeads® EpithelialEnrich (Dynal Biotech, Oslo, Norway), StemSep™ (StemCell Technologies,Inc., Vancouver, BC), and RosetteSep (StemCell Technologies). A skilledartisan will recognize that other methodologies and kits may also beused to enrich or positively select desired cell populations. Dynabeads®Epithelial Enrich contains magnetic beads coated with mAbs specific fortwo glycoprotein membrane antigens expressed on normal and neoplasticepithelial tissues. The coated beads may be added to a sample and thesample then applied to a magnet, thereby capturing the cells bound tothe beads. The unwanted cells are washed away and the magneticallyisolated cells eluted from the beads and used in further analyses.

RosetteSep can be used to enrich cells directly from a blood sample andconsists of a cocktail of tetrameric antibodies that targets a varietyof unwanted cells and crosslinks them to glycophorin A on red bloodcells (RBC) present in the sample, forming rosettes. When centrifugedover Ficoll, targeted cells pellet along with the free RBC. Thecombination of antibodies in the depletion cocktail determines whichcells will be removed and consequently which cells will be recovered.Antibodies that are available include, but are not limited to: CD2, CD3,CD4, CD5, CD8, CD10, CD11 b, CD14, CD15, CD16, CD19, CD20, CD24, CD25,CD29, CD33, CD34, CD36, CD38, CD41, CD45, CD45RA, CD45RO, CD56, CD66B,CD66e, HLA-DR, IgE, and TCRαβ.

Additionally, it is contemplated in the present invention that mAbsspecific for lung tumor antigens can be generated and used in a similarmanner. For example, mAbs that bind to tumor-specific cell surfaceantigens may be conjugated to magnetic beads, or formulated in atetrameric antibody complex, and used to enrich or positively selectmetastatic lung tumor cells from a sample. Once a sample is enriched orpositively selected, cells may be lysed and RNA isolated. RNA may thenbe subjected to RT-PCR analysis using lung tumor-specific primers in areal-time PCR assay as described herein. One skilled in the art willrecognize that enriched or selected populations of cells may be analyzedby other methods (e.g. in situ hybridization or flow cytometry).

In another embodiment, the compositions described herein may be used asmarkers for the progression of cancer. In this embodiment, assays asdescribed above for the diagnosis of a cancer may be performed overtime, and the change in the level of reactive polypeptide(s) orpolynucleotide(s) evaluated. For example, the assays may be performedevery 24-72 hours for a period of 6 months to 1 year, and thereafterperformed as needed. In general, a cancer is progressing in thosepatients in whom the level of polypeptide or polynucleotide detectedincreases over time. In contrast, the cancer is not progressing when thelevel of reactive polypeptide or polynucleotide either remains constantor decreases with time.

Certain in vivo diagnostic assays may be performed directly on a tumor.One such assay involves contacting tumor cells with a binding agent. Thebound binding agent may then be detected directly or indirectly via areporter group. Such binding agents may also be used in histologicalapplications. Alternatively, polynucleotide probes may be used withinsuch applications.

As noted above, to improve sensitivity, multiple tumor protein markersmay be assayed within a given sample. It will be apparent that bindingagents specific for different proteins provided herein may be combinedwithin a single assay. Further, multiple primers or probes may be usedconcurrently. The selection of tumor protein markers may be based onroutine experiments to determine combinations that results in optimalsensitivity. In addition, or alternatively, assays for tumor proteinsprovided herein may be combined with assays for other known tumorantigens.

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a tumor protein. Such antibodies orfragments may be provided attached to a support material, as describedabove. One or more additional containers may enclose elements, such asreagents or buffers, to be used in the assay. Such kits may also, oralternatively, contain a detection reagent as described above thatcontains a reporter group suitable for direct or indirect detection ofantibody binding.

Alternatively, a kit may be designed to detect the level of mRNAencoding a tumor protein in a biological sample. Such kits generallycomprise at least one oligonucleotide probe or primer, as describedabove, that hybridizes to a polynucleotide encoding a tumor protein.Such an oligonucleotide may be used, for example, within a PCR orhybridization assay. Additional components that may be present withinsuch kits include a second oligonucleotide and/or a diagnostic reagentor container to facilitate the detection of a polynucleotide encoding atumor protein.

The following examples are offered by way of illustration and not by wayof limitation.

Examples Example 1 ISOLATION AND CHARACTERIZATION OF CDNA SEQUENCESENCODING LUNG TUMOR POLYPEPTIDES

This example illustrates the isolation of cDNA molecules encoding lungtumor-specific polypeptides from lung tumor cDNA libraries.

A. Isolation of cDNA Sequences from a Lung Squamous Cell CarcinomaLibrary

A human lung squamous cell carcinoma cDNA expression library wasconstructed from poly A⁺ RNA from a pool of two patient tissues using aSuperscript Plasmid System for cDNA Synthesis and Plasmid Cloning kit(BRL Life Technologies, Gaithersburg, Md.) following the manufacturer'sprotocol. Specifically, lung carcinoma tissues were homogenized withpolytron (Kinematica, Switzerland) and total RNA was extracted usingTrizol reagent (BRL Life Technologies) as directed by the manufacturer.The poly A⁺ RNA was then purified using an oligo dT cellulose column asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989.First-strand cDNA was synthesized using the NotI/Oligo-dT18 primer.Double-stranded cDNA was synthesized, ligated with BstXI/EcoRI adaptors(Invitrogen, San Diego, Calif.) and digested with NotI. Following sizefractionation with cDNA size fractionation columns (BRL LifeTechnologies), the cDNA was ligated into the BstXI/NotI site of pcDNA3.1(Invitrogen) and transformed into ElectroMax E. coli DH10B cells (BRLLife Technologies) by electroporation.

Using the same procedure, a normal human lung cDNA expression librarywas prepared from a pool of four tissue specimens. The cDNA librarieswere characterized by determining the number of independent colonies,the percentage of clones that carried insert, the average insert sizeand by sequence analysis. The lung squamous cell carcinoma librarycontained 2.7×10⁶ independent colonies, with 100% of clones having aninsert and the average insert size being 2100 base pairs. The normallung cDNA library contained 1.4×10⁶ independent colonies, with 90% ofclones having inserts and the average insert size being 1800 base pairs.For both libraries, sequence analysis showed that the majority of cloneshad a full length cDNA sequence and were synthesized from mRNA

cDNA library subtraction was performed using the above lung squamouscell carcinoma and normal lung cDNA libraries, as described by Hara etal. (Blood, 84:189-199, 1994) with some modifications. Specifically, alung squamous cell carcinoma-specific subtracted cDNA library wasgenerated as follows. Normal tissue cDNA library (80 μg) was digestedwith BamHI and XhoI, followed by a filling-in reaction with DNApolymerase Klenow fragment. After phenol-chloroform extraction andethanol precipitation, the DNA was dissolved in 133 μl of H₂O,heat-denatured and mixed with 133 μl (133 μg) of Photoprobe biotin(Vector Laboratories, Burlingame, Calif.). As recommended by themanufacturer, the resulting mixture was irradiated with a 270 W sunlampon ice for 20 minutes. Additional Photoprobe biotin (67 μl) was addedand the biotinylation reaction was repeated. After extraction withbutanol five times, the DNA was ethanol-precipitated and dissolved in 23μl H₂O to form the driver DNA.

To form the tracer DNA, 10 μg lung squamous cell carcinoma cDNA librarywas digested with NotI and SpeI, phenol chloroform extracted and passedthrough Chroma spin-400 columns (Clontech, Palo Alto, Calif.).Typically, 5 μg of cDNA was recovered after the sizing column. Followingethanol precipitation, the tracer DNA was dissolved in 5 μl H₂O. TracerDNA was mixed with 15 μl driver DNA and 20 μl of 2× hybridization buffer(1.5 M NaCl/10 mM EDTA/50 mM HEPES pH 7.5/0.2% sodium dodecyl sulfate),overlaid with mineral oil, and heat-denatured completely. The sample wasimmediately transferred into a 68° C. water bath and incubated for 20hours (long hybridization [LH]). The reaction mixture was then subjectedto a streptavidin treatment followed by phenol/chloroform extraction.This process was repeated three more times. Subtracted DNA wasprecipitated, dissolved in 12 μl H₂O, mixed with 8 μl driver DNA and 20μl of 2× hybridization buffer, and subjected to a hybridization at 68°C. for 2 hours (short hybridization [SH]). After removal of biotinylateddouble-stranded DNA, subtracted cDNA was ligated into NotI/SpeI site ofchloramphenicol resistant pBCSK⁺ (Stratagene, La Jolla, Calif.) andtransformed into ElectroMax E. coli DH10B cells by electroporation togenerate a lung squamous cell carcinoma specific subtracted cDNA library(herein after referred to as “lung subtraction I”).

A second lung squamous cell carcinoma specific subtracted cDNA library(referred to as “lung subtraction II”) was generated in a similar way tothe lung subtraction library I, except that eight frequently recoveredgenes from lung subtraction I were included in the driver DNA, and24,000 independent clones were recovered.

To analyze the subtracted cDNA libraries, plasmid DNA was prepared from320 independent clones, randomly picked from the subtracted lungsquamous cell carcinoma specific libraries. Representative cDNA cloneswere further characterized by DNA sequencing with a Perkin Elmer/AppliedBiosystems Division Automated Sequencer Model 373A and/or Model 377(Foster City, Calif.). The cDNA sequences for sixty isolated clones areprovided in SEQ ID NO: 1-60. These sequences were compared to knownsequences in the gene bank using the EMBL and GenBank databases (release96). No significant homologies were found to the sequences provided inSEQ ID NO: 2, 3, 19, 38 and 46. The sequences of SEQ ID NO: 1, 6-8,10-13, 15, 17, 18, 20-27, 29, 30, 32, 34-37, 39-45, 47-49, 51, 52, 54,55 and 57-59 were found to show some homology to previously identifiedexpressed sequence tags (ESTs). The sequences of SEQ ID NO: 9, 28, 31and 33 were found to show some homology to previously identifiednon-human gene sequences and the sequences of SEQ ID NO: 4, 5, 14, 50,53, 56 and 60 were found to show some homology to gene sequencespreviously identified in humans.

The subtraction procedure described above was repeated using the abovelung squamous cell carcinoma cDNA library as the tracer DNA, and theabove normal lung tissue cDNA library and a cDNA library from normalliver and heart (constructed from a pool of one sample of each tissue asdescribed above), plus twenty other cDNA clones that were frequentlyrecovered in lung subtractions I and II, as the driver DNA (lungsubtraction III). The normal liver and heart cDNA library contained1.76×10⁶ independent colonies, with 100% of clones having inserts andthe average insert size being 1600 base pairs. Ten additional cloneswere isolated (SEQ ID NO: 61-70). Comparison of these cDNA sequenceswith those in the gene bank as described above, revealed no significanthomologies to the sequences provided in SEQ ID NO: 62 and 67. Thesequences of SEQ ID NO: 61, 63-66, 68 and 69 were found to show somehomology to previously isolated ESTs and the sequence provided in SEQ IDNO: 70 was found to show some homology to a previously identified ratgene.

In further studies, the subtraction procedure described above wasrepeated using the above lung squamous cell carcinoma cDNA library asthe tracer DNA, and a cDNA library from a pool of normal lung, kidney,colon, pancreas, brain, resting PBMC, heart, skin and esophagus as thedriver DNA, with esophagus cDNAs making up one third of the drivermaterial. Since esophagus is enriched in normal epithelial cells,including differentiated squamous cells, this procedure is likely toenrich genes that are tumor specific rather than tissues specific. ThecDNA sequences of 48 clones determined in this subtraction are providedin SEQ ID NO: 177-224. The sequences of SEQ ID NO: 177, 178, 180, 181,183, 187, 192, 195-197, 208, 211, 212, 215, 216, 218 and 219 showed somehomology to previously identified genes. The sequences of SEQ ID NO:179, 182, 184-186, 188-191, 193, 194, 198-207, 209 210, 213, 214, 217,220 and 224 showed some homology to previously determined ESTs. Thesequence of SEQ ID NO: 221-223 showed no homology to any previouslydetermined sequence.

B. Isolation of CDNA Sequences from a Lung Adenocarcinoma Library

A human lung adenocarcinoma cDNA expression library was constructed asdescribed above. The library contained 3.2×10⁶ independent colonies,with 100% of clones having an insert and the average insert size being1500 base pairs. Library subtraction was performed as described aboveusing the normal lung and normal liver and heart cDNA expressionlibraries described above as the driver DNA. Twenty-six hundredindependent clones were recovered.

Initial cDNA sequence analysis from 100 independent clones revealed manyribosomal protein genes. The cDNA sequences for fifteen clones isolatedin this subtraction are provided in SEQ ID NO: 71-86. Comparison ofthese sequences with those in the gene bank as described above revealedno significant homologies to the sequence provided in SEQ ID NO: 84. Thesequences of SEQ ID NO: 71, 73, 74, 77, 78 and 80-82 were found to showsome homology to previously isolated ESTs, and the sequences of SEQ IDNO: 72, 75, 76, 79, 83 and 85 were found to show some homology topreviously identified human genes.

In further studies, a cDNA library (referred to as mets3616A) wasconstructed from a metastatic lung adenocarcinoma. The determined cDNAsequences of 25 clones sequenced at random from this library areprovided in SEQ ID NO: 255-279. The mets3616A cDNA library wassubtracted against a cDNA library prepared from a pool of normal lung,liver, pancreas, skin, kidney, brain and resting PBMC. To increase thespecificity of the subtraction, the driver was spiked with genes thatwere determined to be most abundant in the mets3616A cDNA library, suchas EF1-alpha, integrin-beta and anticoagulant protein PP4, as well aswith cDNAs that were previously found to be differentially expressed insubtracted lung adenocarcinoma cDNA libraries. The determined cDNAsequences of 51 clones isolated from the subtracted library (referred toas mets3616A-S1) are provided in SEQ ID NO: 280-330.

Comparison of the sequences of SEQ ID NO: 255-330 with those in thepublic databases revealed no significant homologies to the sequences ofSEQ ID NO: 255-258, 260, 262-264, 270, 272, 275, 276, 279, 281, 287,291, 296, 300 and 310. The sequences of SEQ ID NO: 259, 261, 265-269,271, 273, 274, 277, 278, 282-285, 288-290, 292, 294, 297-299, 301,303-309, 313, 314, 316, 320-324 and 326-330 showed some homology topreviously identified gene sequences, while the sequences of SEQ ID NO:280, 286, 293, 302, 310, 312, 315, 317-319 and 325 showed some homologyto previously isolated expressed sequence tags (ESTs).

Example 2 DETERMINATION OF TISSUE SPECIFICITY OF LUNG TUMOR POLYPEPTIDES

Using gene specific primers, mRNA expression levels for sevenrepresentative lung tumor polypeptides described in Example 1 wereexamined in a variety of normal and tumor tissues using RT-PCR.

Briefly, total RNA was extracted from a variety of normal and tumortissues using Trizol reagent as described above. First strand synthesiswas carried out using 2 μg of total RNA with SuperScript II reversetranscriptase (BRL Life Technologies) at 42° C. for one hour. The cDNAwas then amplified by PCR with gene-specific primers. To ensure thesemi-quantitative nature of the RT-PCR, β-actin was used as an internalcontrol for each of the tissues examined. 1 μl of 1:30 dilution of cDNAwas employed to enable the linear range amplification of the β-actintemplate and was sensitive enough to reflect the differences in theinitial copy numbers. Using these conditions, the β-actin levels weredetermined for each reverse transcription reaction from each tissue. DNAcontamination was minimized by DNase treatment and by assuring anegative PCR result when using first strand cDNA that was preparedwithout adding reverse transcriptase.

mRNA Expression levels were examined in five different types of tumortissue (lung squamous cell carcinoma from 3 patients, lungadenocarcinoma, colon tumor from 2 patients, breast tumor and prostatetumor), and thirteen different normal tissues (lung from 4 donors,prostate, brain, kidney, liver, ovary, skeletal muscle, skin, smallintestine, stomach, myocardium, retina and testes). Using a 10-foldamount of cDNA, the antigen LST-S1-90 (SEQ ID NO: 3) was found to beexpressed at high levels in lung squamous cell carcinoma and in breasttumor, and at low to undetectable levels in the other tissues examined.

The antigen LST-S2-68 (SEQ ID NO: 15) appears to be specific to lung andbreast tumor, however, expression was also detected in normal kidney.Antigens LST-S1-169 (SEQ ID NO: 6) and LST-S1-133 (SEQ ID NO: 5) appearto be very abundant in lung tissues (both normal and tumor), with theexpression of these two genes being decreased in most of the normaltissues tested. Both LST-S1-169 and LST-S1-133 were also expressed inbreast and colon tumors. Antigens LST-S1-6 (SEQ ID NO: 7) andLST-S2-I2-5F (SEQ ID NO: 47) did not show tumor or tissue specificexpression, with the expression of LST-S1-28 being rare and onlydetectable in a few tissues. The antigen LST-S3-7 (SEQ ID NO: 63) showedlung and breast tumor specific expression, with its message only beingdetected in normal testes when the PCR was performed for 30 cycles.Lower level expression was detected in some normal tissues when thecycle number was increased to 35. Antigen LST-S3-13 (SEQ ID NO: 66) wasfound to be expressed in 3 out of 4 lung tumors, one breast tumor andboth colon tumor samples. Its expression in normal tissues was lowercompared to tumors, and was only detected in 1 out of 4 normal lungtissues and in normal tissues from kidney, ovary and retina. Expressionof antigens LST-S3-4 (SEQ ID NO: 62) and LST-S3-14 (SEQ ID NO: 67) wasrare and did not show any tissue or tumor specificity. Consistent withNorthern blot analyses, the RT-PCR results on antigen LAT-S1-A-10A (SEQID NO: 78) suggested that its expression is high in lung, colon, stomachand small intestine tissues, including lung and colon tumors, whereasits expression was low or undetectable in other tissues.

A total of 2002 cDNA fragments isolated in lung subtractions I, II andIII, described above, were colony PCR amplified and their mRNAexpression levels in lung tumor, normal lung, and various other normaland tumor tissues were determined using microarray technology (Synteni,Palo Alto, Calif.). Briefly, the PCR amplification products were dottedonto slides in an array format, with each product occupying a uniquelocation in the array. mRNA was extracted from the tissue sample to betested, reverse transcribed, and fluorescent-labeled cDNA probes weregenerated. The microarrays were probed with the labeled cDNA probes, theslides scanned and fluorescence intensity was measured. This intensitycorrelates with the hybridization intensity. Seventeen non-redundantcDNA clones showed over-expression in lung squamous tumors, withexpression in normal tissues tested (lung, skin, lymph node, colon,liver, pancreas, breast, heart, bone marrow, large intestine, kidney,stomach, brain, small intestine, bladder and salivary gland) beingeither undetectable, or 10-fold less compared to lung squamous tumors.The determined cDNA sequences for the clone L513S are provided in SEQ IDNO: 87 and 88; those for L514S are provided in SEQ ID NO: 89 and 90;those for L516S in SEQ ID NO: 91 and 92; that for L517S in SEQ ID NO:93; that for L519S in SEQ ID NO: 94; those for L520S in SEQ ID NO: 95and 96; those for L521S in SEQ ID NO: 97 and 98; that for L522S in SEQID NO: 99; that for L523S in SEQ ID NO: 100; that for L524S in SEQ IDNO: 101; that for L525S in SEQ ID NO: 102; that for L526S in SEQ ID NO:103; that for L527S in SEQ ID NO: 104; that for L528S in SEQ ID NO: 105;that for L529S in SEQ ID NO: 106; and those for L530S in SEQ ID NO: 107and 108. Additionally, the full-length cDNA sequence for L530S isprovided in SEQ ID NO: 151, with the corresponding amino acid sequencebeing provided in SEQ ID NO: 152. L530S shows homology to a splicevariant of a p53 tumor suppressor homologue, p63. The cDNA sequences of7 known isoforms of p63 are provided in SEQ ID NO: 331-337, with thecorresponding amino acid sequences being provided in SEQ ID NO: 338-344,respectively.

Due to polymorphisms, the clone L531S appears to have two forms. A firstdetermined full-length cDNA sequence for L531 S is provided in SEQ IDNO: 109, with the corresponding amino acid sequence being provided inSEQ ID NO:

110. A second determined full-length cDNA sequence for L531S is providedin SEQ ID NO: 111, with the corresponding amino acid sequence beingprovided in SEQ ID NO: 112. The sequence of SEQ ID NO: 111 is identicalto that of SEQ ID NO: 109, except that it contains a 27 by insertion.Similarly, L514S has two alternatively spliced forms; the first variantcDNA is listed as SEQ ID NO: 153, with the corresponding amino acidsequence being provided in SEQ ID NO: 155. The full-length cDNA for thesecond variant form of L514S is provided in SEQ ID NO: 154, with thecorresponding amino acid sequence being provided in SEQ ID NO: 156.

Full length cloning for L524S (SEQ ID NO: 101) yielded two variants (SEQID NO: 163 and 164) with the corresponding amino acid sequences of SEQID NO: 165 and 166, respectively. Both variants have been shown toencode parathyroid hormone-related peptide.

Attempts to isolate the full-length cDNA for L519S, resulted in theisolation of the extended cDNA sequence provided in SEQ ID NO: 173,which contains a potential open reading frame. The amino acid sequenceencoded by the sequence of SEQ ID NO: 173 is provided in SEQ ID NO: 174.Additionally, the full-length cDNA sequence for the clone of SEQ ID NO:100 (known as L523S), a known gene, is provided in SEQ ID NO: 175, withthe corresponding amino acid sequence being provided in SEQ ID NO: 176.In further studies, a full-length cDNA sequence for L523S was isolatedfrom a L523S-positive tumor cDNA library by PCR amplification using genespecific primers designed from the sequence of SEQ ID NO: 175. Thedetermined full-length cDNA sequence is provided in SEQ ID NO: 347. Theamino acid sequence encoded by this sequence is provided in SEQ ID NO:348. This protein sequence differs from the previously published proteinsequence at two amino acid positions, namely at positions 158 and 410.

Comparison of the sequences of L514S and L531S (SEQ ID NO: 87 and 88,and 109, respectively) with those in the gene bank, as described above,revealed no significant homologies to known sequences. The sequences ofL513S, L516S, L517S, L519S, L520S and L530S (SEQ ID NO: 87 and 88, 91and 92, 93, 94, 95 and 96, 107 and 108, respectively) were found to showsome homology to previously identified ESTs. The sequences of L521S,L522S, L523S, L524S, L525S, L526S, L527S, L528S and L529S (SEQ ID NO: 97and 98, 99, 100, 101, 102, 103, 104, 105, and 106, respectively) werefound to represent known genes. The determined full-length cDNA sequencefor L520S is provided in SEQ ID NO: 113, with the corresponding aminoacid sequence being provided in SEQ ID NO: 114. Subsequent microarrayanalysis showed L520S to be overexpressed in breast tumors in additionto lung squamous tumors.

Further analysis demonstrated that L529S (SEQ ID NO: 106 and 115), L525S(SEQ ID NO: 102 and 120) and L527S (SEQ ID NO: 104) are cytoskeletalcomponents and potentially squamous cell specific proteins. L529S isconnexin 26, a gap junction protein. It was found to be highly expressedin one lung squamous tumor, referred to as 9688T, and moderatelyover-expressed in two others. However, lower level expression ofconnexin 26 is also detectable in normal skin, colon, liver and stomach.The over-expression of connexin 26 in some breast tumors has beenreported and a mutated form of L529S may result in over-expression inlung tumors. L525S is plakophilin 1, a desmosomal protein found inplaque-bearing adhering junctions of the skin. Expression levels forL525S mRNA was highly elevated in three out of four lung squamous tumorstested, and in normal skin. L527S has been identified as keratin 6isoform, type II 58 Kd keratin and cytokeratin 13, and showsover-expression in squamous tumors and low expression in normal skin,breast and colon tissues. Keratin and keratin-related genes have beenextensively documented as potential markers for lung cancer includingCYFRA2.1 (Pastor, A., et al, Eur. Respir. J., 10:603-609, 1997). L513S(SEQ ID NO: 87 and 88) shows moderate over-expression in several tumortissues tested, and encodes a protein that was first isolated as apemphigus vulgaris antigen.

L520S (SEQ ID NO: 95 and 96) and L521S (SEQ ID NO: 97 and 98) are highlyexpressed in lung squamous tumors, with L520S being up-regulated innormal salivary gland and L521S being over-expressed in normal skin.Both belong to a family of small proline rich proteins and representmarkers for fully differentiated squamous cells. L521S has beendescribed as a specific marker for lung squamous tumor (Hu, R., et al,Lung Cancer, 20:25-30, 1998). L515S (SEQ ID NO: 162) encodes IGF-β2 andL516S is an aldose reductase homologue. Both are moderately expressed inlung squamous tumors and in normal colon. Notably, L516S (SEQ ID NO: 91and 92) is up-regulated in metastatic tumors but not primary lungadenocarcinoma, an indication of its potential role in metatasis and apotential prognostic marker. L522S (SEQ ID NO: 99) is moderatelyover-expressed in lung squamous tumors with minimum expression in normaltissues. L522S has been shown to belong to a class IV alcoholdehydrogenase, ADH7, and its expression profile suggests it is asquamous cell specific antigen. L523S (SEQ ID NO: 100) is moderatelyover-expressed in lung squamous tumor, human pancreatic cancer celllines and pancreatic cancer tissues, suggesting this gene may be ashared antigen between pancreatic and lung squamous cell cancer.

L524S (SEQ ID NO: 101) is over-expressed in the majority of squamoustumors tested and is homologous with parathyroid hormone-related peptide(PTHrP), which is best known to cause humoral hypercalcaemia associatedwith malignant tumors such as leukemia, prostate and breast cancer. Itis also believed that PTHrP is most commonly associated with squamouscarcinoma of lung and rarely with lung adenocarcinoma (Davidson, L. A.,et al, J. Pathol., 178: 398-401, 1996). L528S (SEQ ID NO: 105) is highlyover-expressed in two lung squamous tumors with moderate expression intwo other squamous tumors, one lung adenocarcinoma and some normaltissues, including skin, lymph nodes, heart, stomach and lung. Itencodes the NMB gene that is similar to the precursor of melanocytespecific gene Pmel17, which is reported to be preferentially expressedin low-metastatic potential melanoma cell lines. This suggests thatL528S may be a shared antigen in both melanoma and lung squamous cellcarcinoma. L526S (SEQ ID NO: 103) was overexpressed in all lung squamouscell tumor tissues tested and has been shown to share homology with agene (ATM) in which a mutation causes ataxia telangiectasia, a geneticdisorder in humans causing a predisposition to cancer, among othersymptoms. ATM encodes a protein that activates a p53 mediated cell-cyclecheckpoint through direct binding and phosphorylation of the p53molecule. Approximately 40% of lung cancers are associated with p53mutations, and it is speculated that over-expression of ATM is a resultof compensation for loss of p53 function, but it is unknown whetherover-expression is the cause of result of lung squamous cell carcinoma.Additionally, expression of L526S (ATM) is also detected in a metastaticbut not lung adenocarcinoma, suggesting a role in metastasis.

Expression of L523S (SEQ ID NO: 175), was examined by real time RT-PCRas described above. In a first study using a panel of lung squamoustumors, L523S was found to be expressed in 4/7 lung squamous tumors, 2/3head and neck squamous tumors and 2/2 lung adenocarcinomas, with lowlevel expression being observed in skeletal muscle, soft palate andtonsil. In a second study using a lung adenocarcinoma panel, expressionof L523S was observed in 4/9 primary adenocarcinomas, 2/2 lung pleuraleffusions, 1/1 metastatic lung adenocarcinomas and 2/2 lung squamoustumors, with little expression being observed in normal tissues.

Expression of L523S in lung tumors and various normal tissues was alsoexamined by Northern blot analysis, using standard techniques. In afirst study, L523S was found to be expressed in a number of lungadenocarcinomas and squamous cell carcinomas, as well as normal tonsil.No expression was observed in normal lung. In a second study using anormal tissue blot (referred to as HB-12) from Clontech, no expressionwas observed in brain, skeletal muscle, colon, thymus, spleen, kidney,liver, small intestine, lung or PBMC, although there was strongexpression in placenta.

Example 3 ISOLATION AND CHARACTERIZATION OF LUNG TUMOR POLYPEPTIDES BYPCR-BASED SUBTRACTION

Eight hundred and fifty seven clones from a cDNA subtraction library,containing cDNA from a pool of two human lung squamous tumors subtractedagainst eight normal human tissue cDNAs including lung, PBMC, brain,heart, kidney, liver, pancreas, and skin, (Clontech, Palo Alto, Calif.)were derived and submitted to a first round of PCR amplification. Thislibrary was subjected to a second round of PCR amplification, followingthe manufacturer's protocol. The resulting cDNA fragments were subclonedinto the P7-Adv vector (Clontech, Palo Alto, Calif.) and transformedinto DH5a E. coli (Gibco, BRL). DNA was isolated from independent clonesand sequenced using a Perkin Elmer/Applied Biosystems Division AutomatedSequencer Model 373A.

One hundred and sixty two positive clones were sequenced. Comparison ofthe DNA sequences of these clones with those in the EMBL and GenBankdatabases, as described above, revealed no significant homologies to 13of these clones, hereinafter referred to as Contigs 13, 16, 17, 19, 22,24, 29, 47, 49, 56-59. The determined cDNA sequences for these clonesare provided in SEQ ID NO: 125, 127-129, 131-133, 142, 144, 148-150, and157, respectively. Contigs 1, 3-5, 7-10, 12, 11, 15, 20, 31, 33, 38, 39,41, 43, 44, 45, 48, 50, 53, 54 (SEQ ID NO: 115-124, 126, 130, 134-141,143, 145-147, respectively) were found to show some degree of homologyto previously identified DNA sequences. Contig 57 (SEQ ID NO: 149) wasfound to represent the clone L519S (SEQ ID NO: 94) disclosed in U.S.patent application Ser. No. 09/123,912, filed Jul. 27, 1998. To the bestof the inventors' knowledge, none of these sequences have beenpreviously shown to be differentially over-expressed in lung tumors.

mRNA expression levels for representative clones in lung tumor tissues,normal lung tissues (n=4), resting PBMC, salivary gland, heart, stomach,lymph nodes, skeletal muscle, soft palate, small intestine, largeintestine, bronchial, bladder, tonsil, kidney, esophagus, bone marrow,colon, adrenal gland, pancreas, and skin (all derived from human) weredetermined by RT-PCR as described above. Expression levels usingmicroarray technology, as described above, were examined in one sampleof each tissue type unless otherwise indicated.

Contig 3 (SEQ ID NO: 116) was found to be highly expressed in all headand neck squamous cell tumors tested (17/17), and expressed in themajority (8/12) of lung squamous tumors, (high expression in 7/12,moderate in 2/12, and low in 2/12), while showing negative expressionfor 2/4 normal lung tissues and low expression in the remaining twosamples. Contig 3 showed moderate expression in skin and soft palate,and lowered expression levels in resting PBMC, large intestine, salivarygland, tonsil, pancreas, esophagus, and colon. Contig 11 (SEQ ID NO:124) was found to be expressed in all head and neck squamous cell tumorstested (17/17), with high levels of expression being seen in 14/1tumors, and moderately levels of expression being seen in 3/17 tumors.Additionally, high expression was seen in 3/12 lung squamous tumors andmoderate expression in 4/12 lung squamous tumors. Contig 11 was negativefor 3/4 normal lung samples, with the remaining sample having only lowexpression. Contig 11 showed low to moderate reactivity to salivarygland, soft palate, bladder, tonsil, skin, esophagus, and largeintestine. Contig 13 (SEQ ID NO: 125) was found to be expressed in allhead and neck squamous cell tumors tested (17/17), with high expressionin 12/17, and moderate expression in 5/17. Contig 13 was expressed in7/12 lung squamous tumors, with high expression in 4/12 and moderateexpression in three samples. Analysis of normal lung samples showednegative expression for 2/4 and low to moderate expression in theremaining two samples. Contig 13 showed low to moderate reactivity toresting PBMC, salivary gland, bladder, pancreas, tonsil, skin,esophagus, and large intestine, as well as high expression in softpalate. Subsequent full-length cloning efforts revealed that contig 13(also known as L761 P) maps to the 3′ untranslated region of the hSeclOp gene. The full-length sequence for this gene is set forth in SEQ IDNO: 368, and encodes the protein set forth in SEQ ID NO: 369.

Contig 16 (SEQ ID NO: 127) was found to be moderately expressed inseveral head and neck squamous cell tumors (6/17) and one lung squamoustumor, while showing no expression in any normal lung samples tested.Contig 16 showed low reactivity to resting PBMC, large intestine, skin,salivary gland, and soft palate. Contig 17 (SEQ ID NO: 128) was shown tobe expressed in all head and neck squamous cell tumors tested (17/17)(highly expressed in 5/17, and moderately expressed in 12/17).Determination of expression levels in lung squamous tumors showed onetumor sample with high expression and 3/12 with moderate levels. Contig17 was negative for 2/4 normal lung samples, with the remaining sampleshaving only low expression. Additionally, low level expression was foundin esophagus and soft palate. Contig 19 (SEQ ID NO: 129) was found to beexpressed in most head and neck squamous cell tumors tested (11/17);with two samples having high expression levels, 6/17 showing moderateexpression, and low expression being found in 3/17. Testing in lungsquamous tumors revealed only moderate expression in 3/12 samples.Expression levels in 2/4 of normal lung samples were negative, the twoother samples having only low expression. Contig 19 showed lowexpression levels in esophagus, resting PBMC, salivary gland, bladder,soft palate and pancreas.

Contig 22 (SEQ ID NO: 131), was shown to be expressed in most head andneck squamous cell tumors tested (13/17) with high expression in four ofthese samples, moderate expression in 6/17, and low expression in 3/17.Expression levels in lung squamous tumors were found to be moderate tohigh for 3/12 tissues tested, with negative expression in two normallung samples and low expression in two other samples (n=4). Contig 22showed low expression in skin, salivary gland and soft palate.Similarly, Contig 24 (SEQ ID NO: 132) was found to be expressed in mosthead and neck squamous cell tumors tested (13/17) with high expressionin three of these samples, moderate expression in 6/17, and lowexpression in 4/17. Expression levels in lung squamous tumors were foundto be moderate to high for 3/12 tissues tested, with negative expressionfor three normal lung samples and low expression in one sample (n=4).Contig 24 showed low expression in skin, salivary gland and soft palate.Contig 29 (SEQ ID NO: 133) was expressed in nearly all head and necksquamous cell tumors tested (16/17): highly expressed in 4/17,moderately expressed in 11/17, with low expression in one sample. Also,it was moderately expressed in 3/12 lung squamous tumors, while beingnegative for 2/4 normal lung samples. Contig 29 showed low to moderateexpression in large intestine, skin, salivary gland, pancreas, tonsil,heart and soft palate. Contig 47 (SEQ ID NO: 142) was expressed in mosthead and neck squamous cell tumors tested (12/17): moderate expressionin 10/17, and low expression in two samples. In lung squamous tumors, itwas highly expressed in one sample and moderately expressed in twoothers (n=13). Contig 47 was negative for 2/4 normal lung samples, withthe remaining two samples having moderate expression. Also, Contig 47showed moderate expression in large intestine, and pancreas, and lowexpression in skin, salivary gland, soft palate, stomach, bladder,resting PBMC, and tonsil.

Contig 48 (SEQ ID NO: 143) was expressed in all head and neck squamouscell tumors tested (17/17): highly expressed in 8/17 and moderatelyexpressed in 7/17, with low expression in two samples. Expression levelsin lung squamous tumors were high to moderate in three samples (n=13).Contig 48 was negative for one out of four normal lung samples, theremaining showing low or moderate expression. Contig 48 showed moderateexpression in soft palate, large intestine, pancreas, and bladder, andlow expression in esophagus, salivary gland, resting PBMC, and heart.Contig 49 (SEQ ID NO: 144) was expressed at low to moderate levels in6/17 head and neck squamous cell tumors tested. Expression levels inlung squamous tumors were moderate in three samples (n=13). Contig 49was negative for 2/4 normal lung samples, the remaining samples showinglow expression. Moderate expression levels in skin, salivary gland,large intestine, pancreas, bladder and resting PBMC were shown, as wellas low expression in soft palate, lymph nodes, and tonsil. Contig 56(SEQ ID NO: 148) was expressed in low to moderate levels in 3/17 headand neck squamous cell tumors tested, and in lung squamous tumors,showing low to moderate levels in three out of thirteen samples.Notably, low expression levels were detected in one adenocarcinoma lungtumor sample (n=2). Contig 56 was negative for 3/4 normal lung samples,and showed moderate expression levels in only large intestine, and lowexpression in salivary gland, soft palate, pancreas, bladder, andresting PBMC. Contig 58, also known as L769P, (SEQ ID NO: 150) wasexpressed at moderate levels in 11/17 head and neck squamous cell tumorstested and low expression in one additional sample. Expression in lungsquamous tumors showed low to moderate levels in three out of thirteensamples. Contig 58 was negative for 3/4 normal lung samples, with onesample having low expression. Moderate expression levels in skin, largeintestine, and resting PBMC were demonstrated, as well as low expressionin salivary gland, soft palate, pancreas, and bladder. Contig 59 (SEQ IDNO: 157) was expressed in some head, neck, and lung squamous tumors. Lowlevel expression of Contig 59 was also detected in salivary gland andlarge intestine.

The full-length cDNA sequence for Contig 22, also referred to as L763P,is provided in SEQ ID NO: 158, with the corresponding amino acidsequence being provided in SEQ ID NO: 159. Real-time RT-PCR analysis ofL763P revealed that it is highly expressed in 3/4 lung squamous tumorsas well as 4/4 head and neck squamous tumors, with low level expressionbeing observed in normal brain, skin, soft pallet and trachea.Subsequent database searches revealed that the sequence of SEQ ID NO:158 contains a mutation, resulting in a frameshift in the correspondingprotein sequence. A second cDNA sequence for L763P is provided in SEQ IDNO: 345, with the corresponding amino acid sequence being provided inSEQ ID NO: 346. The sequences of SEQ ID NO: 159 and 346 are identicalwith the exception of the C-terminal 33 amino acids of SEQ ID NO: 159.

The full-length cDNA sequence incorporating Contigs 17, 19, and 24,referred to as L762P, is provided in SEQ ID NO: 160, with thecorresponding amino acid sequence being provided in SEQ ID NO: 161.Further analysis of L762P has determined it to be a type I membraneprotein and two additional variants have been sequenced. Variant 1 (SEQID NO: 167, with the corresponding amino acid sequence in SEQ ID NO:169) is an alternatively spliced form of SEQ ID NO: 160 resulting indeletion of 503 nucleotides, as well as deletion of a short segment ofthe expressed protein. Variant 2 (SEQ ID NO: 168, with the correspondingamino acid sequence in SEQ ID NO: 170) has a two nucleotide deletion atthe 3′ coding region in comparison to SEQ ID NO: 160, resulting in asecreted form of the expressed protein. Real-time RT-PCR analysis ofL762P revealed that is over-expressed in 3/4 lung squamous tumors and4/4 head & neck tumors, with low level expression being observed innormal skin, soft pallet and trachea.

An epitope of L762P was identified as having the sequenceKPGHWTYTLNNTHHSLQALK (SEQ ID NO: 382), which corresponds to amino acids571-590 of SEQ ID NO:161.

The full-length cDNA sequence for contig 56 (SEQ ID NO: 148), alsoreferred to as L773P, is provided in SEQ ID NO: 171, with the amino acidsequence in SEQ ID NO: 172. L773P was found to be identical todihydroxyl dehydrogenase at the 3′ portion of the gene, with divergent5′ sequence. As a result, the 69 N-terminal amino acids are unique. ThecDNA sequence encoding the 69 N-terminal amino acids is provided in SEQID NO: 349, with the N-terminal amino acid sequence being provided inSEQ ID NO: 350. Real-time PCR revealed that L773P is highly expressed inlung squamous tumor and lung adenocarcinoma, with no detectableexpression in normal tissues. Subsequent Northern blot analysis of L773Pdemonstrated that this transcript is differentially over-expressed insquamous tumors and detected at approximately 1.6 Kb in primary lungtumor tissue and approximately 1.3 Kb in primary head and neck tumortissue.

Subsequent microarray analysis has shown Contig 58, also referred to asL7695 (SEQ ID NO: 150), to be overexpressed in breast tumors in additionto lung squamous tumors.

Example 4 ISOLATION AND CHARACTERIZATION OF LUNG TUMOR POLYPEPTIDES BYPCR-BASED SUBTRACTION

Seven hundred and sixty clones from a cDNA subtraction library,containing cDNA from a pool of two human lung primary adenocarcinomassubtracted against a pool of nine normal human tissue cDNAs includingskin, colon, lung, esophagus, brain, kidney, spleen, pancreas and liver,(Clontech, Palo Alto, Calif.) were derived and submitted to a firstround of PCR amplification. This library (referred to as ALT-1) wassubjected to a second round of PCR amplification, following themanufacturer's protocol. The expression levels of these 760 cDNA clonesin lung tumor, normal lung, and various other normal and tumor tissues,were examined using microarray technology (Incyte, Palo Alto, Calif.).Briefly, the PCR amplification products were dotted onto slides in anarray format, with each product occupying a unique location in thearray. mRNA was extracted from the tissue sample to be tested, reversetranscribed, and fluorescent-labeled cDNA probes were generated. Themicroarrays were probed with the labeled cDNA probes, the slides scannedand fluorescence intensity was measured. This intensity correlates withthe hybridization intensity. A total of 118 clones, of which 55 wereunique, were found to be over-expressed in lung tumor tissue, withexpression in normal tissues tested (lung, skin, lymph node, colon,liver, pancreas, breast, heart, bone marrow, large intestine, kidney,stomach, brain, small intestine, bladder and salivary gland) beingeither undetectable, or at significantly lower levels. One of theseclones, having the sequence as provided in SEQ ID NO:420 (clone #19014),shows homology to a previously identified clone, L773P. Clone L773P hasthe full-length cDNA sequence provided in SEQ ID NO:171 and the aminoacid sequence provided in SEQ ID NO:172 The isolation of clone #19014 isalso described in co-pending U.S. patent application Ser. No.09/285,479, filed Apr. 2, 1999.

Example 5 SYNTHESIS OF POLYPEPTIDES

Polypeptides may be synthesized on a Perkin Elmer/Applied BiosystemsDivision 430A peptide synthesizer using FMOC chemistry with HPTU(O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate)activation. A Gly-Cys-Gly sequence may be attached to the amino terminusof the peptide to provide a method of conjugation, binding to animmobilized surface, or labeling of the peptide. Cleavage of thepeptides from the solid support is carried out using the followingcleavage mixture: trifluoroaceticacid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleavingfor 2 hours, the peptides are precipitated in cold methyl-t-butyl-ether.The peptide pellets are then dissolved in water containing 0.1%trifluoroacetic acid (TFA) and lyophilized prior to purification by C18reverse phase HPLC. A gradient of 0%-60% acetonitrile (containing 0.1%TFA) in water (containing 0.1% TFA) may be used to elute the peptides.Following lyophilization of the pure fractions, the peptides arecharacterized using electrospray or other types of mass spectrometry andby amino acid analysis.

Example 6 PREPARATION OF ANTIBODIES AGAINST LUNG CANCER ANTIGENS

Polyclonal antibodies against the lung cancer antigens L514S, L528S,L531S, L523 and L773P (SEQ ID NO: 155, 225, 112, 176 and 171,respectively) were prepared as follows.

Rabbits were immunized with recombinant protein expressed in andpurified from E. coli as described below. For the initial immunization,400 μg of antigen combined with muramyl dipeptide (MDP) was injectedsubcutaneously (S.C.). Animals were boosted S.C. 4 weeks later with 200μg of antigen mixed with incomplete Freund's Adjuvant (IFA). Subsequentboosts of 100 μg of antigen mixed with IFA were injected S.C. asnecessary to induce high antibody titer responses. Serum bleeds fromimmunized rabbits were tested for antigen-specific reactivity usingELISA assays with purified protein. Polyclonal antibodies against L514S,L528S, L531S, L523S and L773P were affinity purified from high titerpolyclonal sera using purified protein attached to a solid support.

Immunohistochemical analysis using polyclonal antibodies against L514Swas performed on a panel of 5 lung tumor samples, 5 normal lung tissuesamples and normal colon, kidney, liver, brain and bone marrow.Specifically, tissue samples were fixed in formalin solution for 24hours and embedded in paraffin before being sliced into 10 micronsections. Tissue sections were permeabilized and incubated with antibodyfor 1 hr. HRP-labeled anti-mouse followed by incubation with DABchromogen was used to visualize L514S immunoreactivity. L514S was foundto be highly expressed in lung tumor tissue with little or no expressionbeing observed in normal lung, brain or bone marrow. Light staining wasobserved in colon (epithelial crypt cells positive) and kidney (tubulespositive). Staining was seen in normal liver but no mRNA has beendetected in this tissue making this result suspect.

Using the same procedure, immunohistochemical analysis using polyclonalantibodies against L528S demonstrated staining in lung tumor and normallung samples, light staining in colon and kidney, and no staining inliver and heart.

Immunohistochemical analysis using polyclonal antibodies against L531Sdemonstrated staining in lung tumor samples, light membrane staining inmost normal lung samples, epithelial staining in colon, tubule stainingin kidney, ductal epithelial staining in liver and no staining in heart.

Immunohistochemical analysis using polyclonal antibodies against L523Sdemonstrated staining in all lung cancer samples tested but no stainingin normal lung, kidney, liver, colon, bone marrow or cerebellum.

Generation of polyclonal anti-sera against L762P (SEQ ID NO: 169 and170) was performed as follows. 400 micrograms of lung antigen wascombined with 100 micrograms of muramyldipeptide (MDP). An equal volumeof Incomplete Freund's Adjuvant (IFA) was added and then mixed until anemulsion was formed. Rabbits were injected subcutaneously (S.C.). Afterfour weeks the animals were injected S.C. with 200 micrograms of antigenmixed with an equal volume of IFA. Every four weeks animals were boostedwith 100 micrograms of antigen. Seven days following each boost theanimal was bled. Sera was generated by incubating the blood at 4° C. for12-24 hours followed by centrifugation.

Characterization of polyclonal antisera was carried out as follows.Ninety-six well plates were coated with antigen by incubing with 50microliters (typically 1 microgram) at 4° C. for 20 hrs. 250 microlitersof BSA blocking buffer was added to the wells and incubated at roomtemperature for 2 hrs. Plates were washed 6 times with PBS/0.01% Tween.Rabbit sera was diluted in PBSand 50 microliters of diluted sera wasadded to each well and incubated at room temperature for 30 min. Plateswere washed as described above before addition of 50 microliters of goatanti-rabbit horse radish peroxidase (HRP) at a 1:10000 dilution andincubation at room temperature for 30 min. Plates were washed asdescribed above and 100 μl of TMB Microwell Peroxidase Substrate wasadded to each well. Following a 15 minute incubation in the dark at roomtemperature, the colorimetric reaction was stopped with 100 μl 1N H₂SO₄and read immediately at 450 nm. Antisera showed strong reactivity toantigen L762P.

Immunohistochemical analysis using polyclonal antibodies against L762Pdemonstrated staining in all lung cancer samples tested, some lightstaining in the bronchiole epithelium of normal lung, tubule staining inkidney, light epithelial staining in colon and no staining in heart orliver.

In order to evaluate L773P protein expression in various tissues,immunohistochemistry (IHC) analysis was performed using an affinitypurified L773P polyclonal antibody. Briefly, tissue samples were fixedin formalin solution for 12-24 hrs and embedded in paraffin before beingsliced into 8 micron sections. Steam heat induced epitope retrieval(SHIER) in 0.1 M sodiuym citrate buffer (pH 6.0) was used for optimalstaining conditions. Sections were incubated with 10% serum/PBS for 5minutes. Primary antibody was added to each section for 25 minutes atindicated concentrations followed by 25 minute incubation with eitheranti-rabbit or anti-mouse biotinylated antibody. Endogenous peroxidaseactivitiy was blocked by three 1.5 minute incubations with hydrogenperoxidase. The avidin biotin complex/horse radish peroxidase (ABC/HRP)system was used along with DAB chromogen to visualize L773P expression.Slides were counterstainied with hematoxylin to visualize cell nuclei.Using this approach, L773P protein was detected in 6/8 lung tumors, 4/6normal lung samples (very light staining in some cases), 1/1 kidneysamples (very light staining), 0/1 heart samples, 1/1 colon samples(very light staining) and 0/1 liver samples.

Example 7 PEPTIDE PRIMING OF MICE AND PROPAGATION OF CTL LINES

Immunogenic peptides from the lung cancer antigen L762P (SEQ ID NO: 161)for HLA-A2/K^(b)-restricted CD8+ T cells were identified as follows.

The location of HLA-A2 binding peptides within the lung cancer antigenL762P (SEQ ID NO: 161) was predicted using a computer program whichpredicts peptides sequences likely to being to HLA-A*0201 by fitting tothe known peptide binding motif for HLA-A*0201 (Rupert et al. (1993)Cell 74:929; Rammensee et al. (1995) Immunogenetics 41:178-228). Aseries of 19 synthetic peptides corresponding to a selected subset ofthe predicted HLA-A*0201 binding peptides was prepared as describedabove.

Mice expressing the transgene for human HLA A2/K^(b) (provided by Dr L.Sherman, The Scripps Research Institute, La Jolla, Calif.) wereimmunized with the synthetic peptides, as described by Theobald et al.,Proc. Natl. Acad. Sci. USA 92:11993-11997, 1995, with the followingmodifications. Mice were immunized with 50 μg of L726P peptide and 120μg of an I-A^(b) binding peptide derived from hepatitis B virus proteinemulsified in incomplete Freund's adjuvant. Three weeks later these micewere sacrificed and single cell suspensions prepared. Cells were thenresuspended at 7×10⁶ cells/ml in complete media (RPMI-1640; Gibco BRL,Gaithersburg, Md.) containing 10% FCS, 2 mM Glutamine (Gibco BRL),sodium pyruvate (Gibco BRL), non-essential amino acids (Gibco BRL),2×10⁻⁵ M 2-mercaptoethanol, 50 U/ml penicillin and streptomycin, andcultured in the presence of irradiated (3000 rads) L762P peptide- (5μg/ml) and 10mg/ml B₂-microglobulin-(3 μ/ml) LPS blasts (A2 transgenicspleens cells cultured in the presence of 7 μg/ml dextran sulfate and 25μg/ml LPS for 3 days). After six days, cells (5×10⁵/ml) wererestimulated with 2.5×10⁶/ml peptide-pulsed irradiated (20,000 rads)EL4A2Kb cells (Sherman et al, Science 258:815-818, 1992) and 5×10⁶/mlirradiated (3000 rads) A2/K^(b)-transgenic spleen feeder cells. Cellswere cultured in the presence of 10 U/ml IL-2. Cells were restimulatedon a weekly basis as described, in preparation for cloning the line.

Peptide-specific cell lines were cloned by limiting dilution analysiswith irradiated (20,000 rads) L762P peptide-pulsed EL4 A2Kb tumor cells(1×10⁴ cells/well) as stimulators and irradiated (3000 rads)A2/K^(b)-transgenic spleen cells as feeders (5×10⁵ cells/well) grown inthe presence of 10 U/ml IL-2. On day 7, cells were restimulated asbefore. On day 14, clones that were growing were isolated and maintainedin culture.

Cell lines specific for the peptides L762P-87 (SEQ ID NO: 226;corresponding to amino acids 87-95 of SEQ ID NO: 161), L762P-145 (SEQ IDNO: 227; corresponding to amino acids 145-153 of SEQ ID NO: 161),L762P-585 (SEQ ID NO: 228; corresponding to amino acids 585-593 of SEQID NO: 161), L762P-425 (SEQ ID NO: 229; corresponding to amino acids425-433 of SEQ ID NO: 161), L762P(10)-424 (SEQ ID NO: 230; correspondingto amino acids 424-433 of SEQ ID NO: 161) and L762P(10)-458 (SEQ ID NO:231; corresponding to amino acids 458-467 of SEQ ID NO: 161)demonstrated significantly higher reactivity (as measured by percentspecific lysis) against L762P peptide-pulsed EL4-A2/K^(b) tumor targetcells than control peptide-pulsed EL4-A2/K^(b) tumor target cells.

Example 8 IDENTIFICATION OF CD4 IMMUNOGENIC T CELL EPITOPES DERIVED FROMTHE LUNG CANCER ANTIGEN L762P

CD4 T cell lines specific for the antigen L762P (SEQ ID NO: 161) weregenerated as follows.

A series of 28 overlapping peptides were synthesized that spannedapproximately 50% of the L762P sequence. For priming, peptides werecombined into pools of 4-5 peptides, pulsed at 20 micrograms/ml intodendritic cells for 24 hours. The dendritic cells were then washed andmixed with positively selected CD4+ T cells in 96 well U-bottomedplates. Forty cultures were generated for each peptide pool. Cultureswere restimulated weekly with fresh dendritic cells loaded with peptidepools. Following a total of 3 stimulation cycles, cells were rested foran additional week and tested for specificity to antigen presentingcells (APC) pulsed with peptide pools using interferon-gamma ELISA andproliferation assays. For these assays, adherent monocytes loaded witheither the relevant peptide pool or an irrelevant peptide were used asAPC. T cell lines that appeared to specifically recognize L762P peptidepools both by cytokine release and proliferation were identified foreach pool. Emphasis was placed on identifying T cells with proliferativeresponses. T cell lines that demonstrated either both L762P-specificcytokine secretion and proliferation, or strong proliferation alone werefurther expanded to be tested for recognition of individual peptidesfrom the pools, as well as for recognition of recombinant L762P. Thesource of recombinant L762P was E. coli, and the material was partiallypurified and endotoxin positive. These studies employed 10 micrograms ofindividual peptides, 10 or 2 micrograms of an irrelevant peptide, and 2or 0.5 micrograms of either

L762P protein or an irrelevant, equally impure, E. coli generatedrecombinant protein. Significant interferon-gamma production and CD4 Tcell proliferation was induced by a number of L762P-derived peptides ineach pool. The amino acid sequences for these peptides are provided inSEQ ID NO: 232-251. These peptides correspond to amino acids 661-680,676-696, 526-545, 874-893, 811-830, 871-891, 856-875, 826-845, 795-815,736-755, 706-725, 706-725, 691-710, 601-620, 571-590, 556-575, 616-635,646-665, 631-650, 541-560 and 586-605, respectively, of SEQ ID NO: 161.

CD4 T cell lines that demonstrated specificity for individualL762P-derived peptides were further expanded by stimulation with therelevant peptide at 10 micrograms/ml. Two weeks post-stimulation, T celllines were tested using both proliferation and IFN-gamma ELISA assaysfor recognition of the specific peptide. A number of previouslyidentified T cells continued to demonstrate L762P-peptide specificactivity. Each of these lines was further expanded on the relevantpeptide and, following two weeks of expansion, tested for specificrecognition of the L762P-peptide in titration experiments, as well asfor recognition of recombinant E. coli-derived L762P protein. For theseexperiments, autologous adherent monocytes were pulsed with either therelevant L762P-derived peptide, an irrelevant mammaglobin-derivedpeptide, recombinant E. coli-derived L762P (approx. 50% pure), or anirrelevant E. coli-derived protein. The majority of T cell lines werefound to show low affinity for the relevant peptide, since specificproliferation and IFN-gamma ratios dramatically decreased as L762Ppeptide was diluted. However, four lines were identified thatdemonstrated significant activity even at 0.1 micrograms/ml peptide.Each of these lines (referred to as A/D5, D/F5, E/A7 and E/B6) alsoappeared to specifically proliferate in response to the E. coli-derivedL762P protein preparation, but not in response to the irrelevant proteinpreparation. The amino acid sequences of the L762P-derived peptidesrecognized by these lines are provided in SEQ ID NO: 234, 249, 236 and245, respectively. No protein specific IFN-gamma was detected for any ofthe lines. Lines A/D5, E/A7 and E/B6 were cloned on autologous adherentmonocytes pulsed with the relevant peptide at 0.1 (A/D5 and E/A7) or 1(D/F5) microgram/ml. Following growth, clones were tested forspecificity for the relevant peptide. Numerous clones specific for therelevant peptide were identified for lines ND5 and E/A7.

Example 9 PROTEIN EXPRESSION OF LUNG TUMOR-SPECIFIC ANTIGENS

A. Expression of L514S in E. coli

The lung tumor antigen L514S (SEQ ID NO: 89) was subcloned into theexpression vector pE32b at NcoI and NotI sites, and transformed into E.coli using standard techniques. The protein was expressed from residues3-153 of SEQ ID NO: 89. The expressed amino acid sequence and thecorresponding DNA sequence are provided in SEQ ID NO: 252 and 253,respectively.

B. Expression of L762P

Amino acids 32-944 of the lung tumor antigen L762P (SEQ ID NO: 161),with a 6× His Tag, were subcloned into a modified pET28 expressionvector, using kanamycin resistance, and transformed into BL21 CodonPlususing standard techniques. Low to moderate levels of expression wereobserved. The determined DNA sequence of the L762P expression constructis provided in SEQ ID NO: 254.

Example 10 IDENTIFICATION OF MHC CLASS II RESTRICTING ALLELE FOR L762PPEPTIDE-SPECIFIC RESPONSES

A panel of HLA mismatched antigen presenting cells (APC) were used toidentify the MHC class II restricting allele for the L762P-peptidespecific responses of CD4 T cell clones derived from lines thatrecognized L762P peptide and recombinant protein. Clones from two lines,AD-5 and EA-7, were tested as described below. The AD-5 derived cloneswere found to be restricted by the HLA-DRB-1101 allele, and an EA-7derived clone was found to be restricted by the HLA DRB-0701 orDQB1-0202 allele. Identification of the restriction allele allowstargeting of vaccine therapies using the defined peptide to individualsthat express the relevant class II allele. Knowing the relevantrestricting allele will also enable clinical monitoring for responses tothe defined peptide since only individuals that express the relevantallele will be monitored.

CD4 T cell clones derived from line AD-5 and EA-7 were stimulated onautologous APC pulsed with the specific peptide at 10 μg/ml, and testedfor recognition of autologous APC (from donor D72) as well as against apanel of APC partially matched with D72 at class II alleles. Table 2shows the HLA class typing of the APC tested. Adherent monocytes(generated by 2 hour adherence) from four different donors, referred toas D45, D187, D208, and D326, were used as APC in these experiments.Autologous APC were not included in the experiment. Each of the APC werepulsed with the relevant peptide (5a for AD-5 and 3e for 3A-7) or theirrelevant mammoglobin peptide at 10 μg/ml, and cultures wereestablished for 10,000 T cells and about 20,000 APC/well. As shown inTable 3, specific proliferation and cytokine production could bedetected only when partially matched donor cells were used as APC. Basedon the MHC typing analysis, these results strongly suggest that therestricting allele for the L762-specific response of the AD-5 derivedclones is HLA-DRB-1101 and for the EA-7 derived clone the restrictingallele is HLA DRB-0701 or DQB1-0202.

TABLE 2 HLA Typing of APC DONOR DR DR DQ DQ D72 B1-1101 B1-0701 B1-0202B1-0301 D45 −3 −15 B1-0201 B1-0602 D187 −4 −15 −1 −7 D208 B1-1101B1-0407 −3 −3 D326 B1-0301 B1-0701 B1-0202 B1-0201

TABLE 3 L762P Peptide Responses Map to HLA DR Alleles AD-5 A11 B10 C10C11 E6 F1 γ- γ- γ- γ- γ- γ- Donor Prol IFN Prol IFN Prol IFN Prol IFNProl IFN Prol IFN D72 46 31 34 24 31 40 DR-0701, -1101, DQ-0202, -7 D453.2 1.7 5.5 1.2 3.3 1 1.0 1.5 1.1 1.1 1.6 1.1 DR-3, -15, DQ-1, -0201D187 1.4 1.2 1.3 1 1.4 1.1 1.4 1.7 1.0 1.1 1.4 1.2 DR-4, -15, DQ-1, -7D208 138 13 38 5.4 18.8 10 14.6 4.6 15.3 6.1 45.9 8.6 DR-4, -1101, DQ-3D326 0.7 4 0.3 1 0.3 1.4 1.0 2 0.8 1.1 0.3 1.1 DR-3, -0701, DQ-0202 AD-5EA-7 F9 G8 G9 G10 G12 γ- γ- γ- γ- γ- Donor Prol IFN Prol IFN Prol IFNProl IFN Prol IFN D72 55 45 43 91 10 DR-0701, -1101, DQ-0202, -7 D45 1.41.3 0.2 1.1 1.1 1.1 1.2 1.5 0.8 1.1 DR-3, -15, DQ-1, -0201 D187 1.2 1.10.9 1 1.0 1 1.0 1.6 0.5 1 DR-4, -15, DQ-1, -7 D208 73.3 14.1 38.0 7.7174.3 16.1 113.6 19.6 0.8 1 DR-4, -1101, DQ-3 D326 0.7 1.1 0.6 1.2 0.4 11.2 5 14.1 6.8 DR-3, -0701, DQ-0202

Example 11 FUSION PROTEINS OF N-TERMINAL AND C-TERMINAL PORTIONS OFL763P

In another embodiment, a Mycobacterium tuberculosis-derivedpolynucleotide, referred to as Ra12, is linked to at least animmunogenic portion of a polynucleotide of this invention. Ra12compositions and methods for their use in enhancing expression ofheterologous polynucleotide sequences are described in U.S. PatentApplication 60/158,585, the disclosure of which is incorporated hereinby reference in its entirety. Briefly, Ra12 refers to a polynucleotideregion that is a subsequence of a Mycobacterium tuberculosis MTB32Anucleic acid. MTB32A is a serine protease of 32 KD molecular weightencoded by a gene in virulent and avirulent strains of M. tuberculosis.The nucleotide sequence and amino acid sequence of MTB32A have beendescribed (for example, U.S. Patent Application 60/158,585; see also,Skeiky et al., Infection and Immun. (1999) 67:3998-4007, incorporatedherein by reference). Surprisingly, it was discovered that a 14 KDC-terminal fragment of the MTB32A coding sequence expresses at highlevels on its own and remains as a soluble protein throughout thepurification process. Moreover, this fragment may enhance theimmunogenicity of heterologous antigenic polypeptides with which it isfused. This 14 KD C-terminal fragment of the MTB32A is referred toherein as Ra12 and represents a fragment comprising some or all of aminoacid residues 192 to 323 of MTB32A.

Recombinant nucleic acids which encode a fusion polypeptide comprising aRa12 polypeptide and a heterologous lung tumor polypeptide of interest,can be readily constructed by conventional genetic engineeringtechniques. Recombinant nucleic acids are constructed so that,preferably, a Ra12 polynucleotide sequence is located 5′ to a selectedheterologous lung tumor polynucleotide sequence. It may also beappropriate to place a Ra12 polynucleotide sequence 3′ to a selectedheterologous polynucleotide sequence or to insert a heterologouspolynucleotide sequence into a site within a Ra12 polynucleotidesequence.

In addition, any suitable polynucleotide that encodes a Ra12 or aportion or other variant thereof can be used in constructing recombinantfusion polynucleotides comprising Ra12 and one or more lung tumorpolynucleotides disclosed herein. Preferred Ra12 polynucleotidesgenerally comprise at least about 15 consecutive nucleotides, at leastabout 30 nucleotides, at least about 60 nucleotides, at least about 100nucleotides, at least about 200 nucleotides, or at least about 300nucleotides that encode a portion of a Ra12 polypeptide.

Ra12 polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a Ra12 polypeptide or a portion thereof) or maycomprise a variant of such a sequence. Ra12 polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the biological activity of the encoded fusionpolypeptide is not substantially diminished, relative to a fusionpolypeptide comprising a native Ra12 polypeptide. Variants preferablyexhibit at least about 70% identity, more preferably at least about 80%identity and most preferably at least about 90% identity to apolynucleotide sequence that encodes a native Ra12 polypeptide or aportion thereof.

Two specific embodiments of fusions between Ra12 and antigens of thepresent invention are described in this example.

A. N-Terminal Portion of L763P

A fusion protein of full-length Ra12 and the N-terminal portion of L763P(referred to as L763P-N; amino acid residues 1-130 of SEQ ID NO: 159)was expressed as a single recombinant protein in E. coli. The cDNA forthe N-terminal portion was obtained by PCR with a cDNA for the fulllength L763P and primers L763F3 (5′ CGGCGAATTCATGGATTGGGGGACGCTGC; SEQID NO: 383) and 1763RV3 (5′ CGGCCTCGAGTCACCCCTCTATCCGAACCTTCTGC; SEQ IDNO: 384). The PCR product with expected size was recovered from agarosegel, digested with restriction enzymes EcoRI and XhoI, and cloned intothe corresponding sites in the expression vector pCRX1. The sequence forthe fusion of full-length of Ra12 and L763P-N was confirmed by DNAsequencing. The determined cDNA sequence is provided in SEQ ID NO:351,with the corresponding amino acid sequence being provided in SEQ ID NO:352).

B. C-Terminal Portion of L763P

A fusion protein of full-length Ra12 and the C-terminal portion of L763P(referred to as L763P-C; amino acid residues 100-262 of SEQ ID NO: 159)was expressed as a single recombinant protein in E. coli. The cDNA ofthe C-terminal portion of L763P was obtained by PCR with a cDNA for thefull length of L763P and primers L763F4 (5′CGGCGAATTCCACGAACCACTCGCAAGTTCAG; SEQ ID NO: 385) and L763RV4 (5′CGGCTCGAG-TTAGCTTGGGCCTGTGATTGC; SEQ ID NO: 386). The PCR product withexpected size was recovered from agarose gel, digested with restrictionenzymes EcoRI and XhoI, and cloned into the corresponding sites in theexpression vector pCRX1. The sequence for the fusion of full-length Ra12and L763P-C was confirmed by DNA sequencing. The determined DNA sequenceis provided in SEQ ID NO:353, with the corresponding amino acid sequencebeing provided in SEQ ID NO: 354.

The recombinant proteins described in this example are useful for thepreparation of vaccines, for antibody therapeutics, and for diagnosis oflung tumors.

Example 12 EXPRESSION IN E. COLI OF L762P HIS TAG FUSION PROTEIN

PCR was performed on the L762P coding region with the following primers:

Forward primer starting at amino acid 32.

PDM-278 5′ggagtacagcttcaagacaatggg 3′ (SEQ ID NO: 355) Tm 57° C.

Reverse primer including natural stop codon after amino acid 920,creating EcoRI site

(SEQ ID NO: 356) PDM-280 5′ccatgggaattcattataataattttgttcc 3′ TM55° C.

The PCR product was digested with EcoRI restriction enzyme, gel purifiedand then cloned into pPDM His, a modified pET28 vector with a His tag inframe, which had been digested with Eco72I and EcoRI restrictionenzymes. The correct construct was confirmed by DNA sequence analysisand then transformed into BL21 (DE3) pLys S and BL21 (DE3) CodonPlus RILexpression hosts.

The protein sequence of expressed recombinant L762P is shown in SEQ IDNO:357, and the DNA sequence is shown in SEQ ID NO:358.

Example 13 EXPRESSION IN E. COLI OF A L773PA HIS TAG FUSION PROTEIN

The L773PA coding region (encoding amino acids 2-71 of SEQ ID NO: 172)was PCR amplified using the following primers:

Forward primer for L773PA starting at amino acid 2:

(SEQ ID NO: 359) PDM-299 5′tggcagcccctcttcttcaagtggc 3′ Tm63° C.

Reverse primer for L773PA creating artificial stop codon after aminoacid 70:

(SEQ ID NO: 360) PDM-355 5′cgccagaattcatcaaacaaatctgttagcacc 3′ Tm62° C.

The resulting PCR product was digested with EcoRI restriction enzyme,gel purified and then cloned into pPDM His, a modified pET28 vector witha His tag in frame, which had been digested with Eco72I and EcoRIrestriction enzymes. The correct construct was confirmed by DNA sequenceanalysis and transformed into BL21 (DE3) pLys S and BL21 (DE3) CodonPlusRIL expression hosts.

The protein sequence of expressed recombinant L773PA is shown in SEQ IDNO:361, and the DNA sequence is shown in SEQ ID NO:362.

Example 14 IDENTIFICATION OF EPITOPES DERIVED FROM LUNG TUMOR SPECIFICPOLYPEPTIDES

A series of peptides from the L773P amino acid sequence (SEQ ID NO: 172)were synthesized and used in in vitro priming experiments to generatepeptide-specific CD4 T cells. These peptides were 20-mers thatoverlapped by 15 amino acids and corresponded to amino acids 1-69 of theL773P protein. This region has been demonstrated to be tumor-specific.Following three in vitro stimulations, CD4 T cell lines were identifiedthat produced IFNγ in response to the stimulating peptide but not thecontrol peptide. Some of these T cell lines demonstrated recognition ofrecombinant L773P and L773PA (tumor-specific region) proteins.

To perform the experiments, a total of eleven 20-mer peptides (SEQ IDNO: 363, 365 and 387-395) overlapping by 15 amino acids and derived fromthe N-terminal tumor-specific region of L773P (corresponding to aminoacids 1-69 of SEQ ID NO:172) were generated by standard procedures.Dendritic cells were derived from PBMC of a normal donor using GMCSF andIL-4 by standard protocol. Purified CD4 T cells were generated from thesame donor as the dendritic cells using MACS beads and negativeselection of PBMCs. Dendritic cells were pulsed overnight with theindividual 20-mer peptides at a concentration of 10 μg/ml. Pulseddendritic cells were washed and plated at 1×10⁴/well of a 96-wellU-bottom plates, and purified CD4 cells were added at 1×10⁵ well.Cultures were supplemented with 10 ng/ml IL-6 and 5 ng/ml IL-12, andincubated at 37° C. Cultures were re-stimulated as above on a weeklybasis using as APC dendritic cells generated and pulsed as above,supplemented with 5 ng/ml IL-7 and 10 μg/ml IL-2. Following 3 in vitrostimulation cycles, cell lines (each corresponding to one well) weretested for cytokine production in response to the stimulating peptidevs. an irrelevant peptide.

A small number of individual CD4 T cell lines (9/528) demonstratedcytokine release (IFNγ) in response to the stimulating peptide but notto control peptide. The CD4 T cell lines that demonstrated specificactivity were restimulated on the appropriate L773P peptide andreassayed using autologous dendritic cells pulsed with 10 μg/ml of theappropriate L773P peptide, an irrelevant control peptide, recombinantL773P protein (amino acids 2-364, made in E. coli), recombinant L773PA(amino acids 2-71, made in E. coli), or an appropriate control protein(L3E, made in E. coli). Three of the nine lines tested (1-3C, 1-6G, and4-12B) recognized the appropriate L773P peptide as well as recombinantL773P and L773PA. Four of the lines tested (4-8A, 4-8E, 4-12D, and4-12E) recognized the appropriate L773P peptide only. Two of the linestested (5-6F and 9-3B) demonstrated non-specific activity.

These results demonstrate that the peptide sequencesMWQPLFFKWLLSCCPGSSQI (amino acids 1-20 of SEQ ID NO: 172; SEQ ID NO:363)and GSSQIAAAASTQPEDDINTQ (amino acids 16-35 of SEQ ID NO: 172; SEQ IDNO: 365) may represent naturally processed epitopes of L773P, which arecapable of stimulating human class II MHC-restricted CD4 T cellresponses.

In subsequent studies, the above epitope mapping experiment was repeatedusing a different donor. Again, some of the resulting T cell lines werefound to respond to peptide and recombinant protein. An additionalpeptide was found to be naturally processed. Specifically, purified CD4cells were stimulated on a total of eleven 20-mer peptides overlappingby 15 amino acids (SEQ ID NO: 363, 387, 388, 365 and 389-395,respectively). The priming was carried out as described above, exceptthat a peptide concentration of 0.5 ug/mL rather than 10 ug/mL wasemployed. In the initial screen of the cell lines 9 of the 528 linesreleased at least a three-fold greater level of IFN-gamma withstimulating peptide vs. control peptide. These 9 lines were restimulatedon the appropriate peptide and then tested on dendritic cells pulsedwith a titration of appropriate peptide (10 ug/mL, 1 ug/mL and 0.1ug/mL), and 10 ug/mL of a control peptide. Six of the 9 lines recognizedrecombinant L773P as well as peptide. The six lines referred to as 1-1E,1-2E, 1-4H, 1-6A, 1-6G and 2-12B recognized L773PA and the appropriatepeptide. These results demonstrate that the peptides of SEQ ID NO: 363and 387 represent naturally processed epitopes of L773P.

Using the procedures described above, CD4+ T cell responses weregenerated from PBMC of normal donors using dendritic cells pulsed withoverlapping 20-mer peptides (SEQ ID NO: 396-419) spanning the L523Spolypeptide sequence (SEQ ID NO: 176). A number of CD4+ T cellsdemonstrated reactivity with the priming peptides as well as with L523Srecombinant protein, with the dominant reactivity of these lines beingwithin the peptides 4, 7 and 21 (SEQ ID NO: 399, 402 and 416;corresponding to amino acids 30-39, 60-79 and 200-219, respectively, ofSEQ ID NO: 176).

Epitopes within the scope of the invention include epitopes restrictedby other class II MHC molecules. In addition, variants of the peptidecan be produced wherein one or more amino acids are altered such thatthere is no effect on the ability of the peptides to bind to MHCmolecules, no effect on their ability to elicit T cell responses, and noeffect on the ability of the elicited T cells to recognize recombinantprotein.

Example 15 SURFACE EXPRESSION OF L762P AND ANTIBODY EPITOPES THEREOF

Rabbits were immunized with full-length histidine-tagged L762P proteingenerated in E. coli. Sera was isolated from rabbits and screened forspecific recognition of L762P in ELISA assays. One polyclonal serum,referred to as 2692L, was identified that specifically recognizedrecombinant L762P protein. The 2692L anti-L762P polyclonal antibodieswere purified from the serum by affinity purification using L762Paffinity columns. Although L762P is expressed in a subset of primarylung tumor samples, expression appears to be lost in established lungtumor cell lines. Therefore, to characterize surface expression ofL762P, a retrovirus construct that expresses L762P was used to transduceprimary human fibroblasts as well as 3 lung tumor cell lines (522-23,HTB, and 343T). Transduced lines were selected and expanded to examineL762P surface expression by FACS analysis. For this analysis,non-transduced and transduced cells were harvested using celldissociation medium, and incubated with 10-50 micrograms/ml of eitheraffinity purified anti-L762P or irrelevant antisera. Following a 30minute incubation on ice, cells were washed and incubated with asecondary, FITC conjugated, anti rabbit IgG antibody as above. Cellswere washed, resuspended in buffer with Propidium Iodide (PI) andexamined by FACS using an Excalibur fluorescence activated cell sorter.For FACS analysis, PI-positive (i.e. dead/permeabilized cells) wereexcluded. The polyclonal anti-L762P sera specifically recognized andbound to the surface of L762P-transduced cells but not thenon-transduced counterparts. These results demonstrate that L762P islocalized to the cell surface of both fibroblasts as well as lung tumorcells.

To identify the peptide epitopes recognized by 2692L, an epitope mappingapproach was pursued. A series of overlapping 19-21 mers (5 amino acidoverlap) was synthesized that spanned the C terminal portion of L762P(amino acids 481-894 of SEQ ID NO: 161). In an initial experimentpeptides were tested in pools. Specific reactivity with the L762Pantiserum was observed with pools A, B, C, and E. To identify thespecific peptides recognized by the antiserum, flat bottom 96 wellmicrotiter plates were coated with individual peptides at 10microgram/ml for 2 hours at 37° C. Wells were then aspirated and blockedwith phosphate buffered saline containing 5% (w/v) milk for 2 hours at37° C., and subsequently washed in PBS containing 0.1% Tween 20 (PBST).Purified rabbit anti-L762P serum 2692L was added at 200 or 20 ng/well totriplicate wells in PBST and incubated overnight at room temperature.This was followed by washing 6 times with PBST and subsequentlyincubating with HRP-conjugated donkey anti rabbit IgG (H+L)AffinipureF(ab') fragment at 1:2,000 for 60 minutes. Plates were then washed, andincubated in tetramethyl benzidine substrate. Reactions were stopped bythe addition of 1N sulfuric acid and plates were read at 450/570 nmusing an ELISA plate reader.

The resulting data, presented in Table 4 below, demonstrates that theL762P antisera recognized at least 6 distinct peptide epitopes from the3′ half of L762P.

TABLE 4 ELISA activity (OD 450-570 200 ng 20 ng Peptide (starting aminopolyclonal polyclonal acid of L762P) pool serum serum A (481) A 1.76 1.0B (495) A 0.14 .06 C (511) E 0.47 0.18 D (526) E 0.11 0.09 E (541) A0.11 0.04 F (556) A 0.04 0.02 G (571) A 0.06 0.02 H (586) B 0.1 0.03 I(601) B 0.25 0.06 J (616) B 0.1 0.03 K (631) E 0.1 0.08 L (646) B 0.280.12 M (661) B 0.14 0.03 N (676) C 0.12 0.1 O (691) C 1.1 0.23 P (706) C0.1 0.03 Q (721) C 0.11 0.05 R (736) E 0.12 0.04 S (751) C 0.15 0.06 U(781) D 0.12 0.06 V (795) F 0.07 0.05 X (826) D 0.1 0.03 Y (841) D 0.170.07 Z (856) D 0.16 0.08 AA (871) F 0.17 0.05 BB (874) F 0.14 0.11 Nopeptide 0.15 0.045

Individual peptides were identified from each of the pools, andadditionally a weak reactivity was identified with peptide BB from poolF. The relevant peptide epitopes are summarized in the Table 5 below.The amino acid sequences for peptides BB, O, L, I, A and C are providedin SEQ ID NO: 376-381, respectively, with the corresponding cDNAsequences being provided in SEQ ID NO: 373, 370, 372, 374, 371 and 375,respectively.

TABLE 5 ELISA activity (OD 450-570) Amino Nucleotides acids of ofPeptide L762P L762P Sequence pool 200 ng 20 ng A 1441-1500 481-500SRISSGTGDIFQQHIQLEST A 1.76 1.0 C 1531-1590 511-530 KNTVTVDNTVGNDTMFLVTWE 0.47 0.18 I 1801-1860 601-620 AVPPATVEAFVERDSLHFPH B 0.25 0.06 L1936-1955 646-665 PETGDPVTLRLLDDGAGADV B 0.28 0.12 O 2071-2130 691-710VNHSPSISTPAHSIPGSHAMIL C 1.1 0.23 BB 2620-2679 874-893LQSAVSNIAQAPLFIPPNSD F 0.14 0.11 None — — — — 0.15 0.05

Example 16 DETECTION OF ANTIBODIES AGAINST LUNG TUMOR ANTIGENS INPATIENT SERA

Antibodies specific for the lung tumor antigens L773PA (SEQ ID NO:361),L514S (SEQ ID NO:155 and 156), L523S (SEQ ID NO:176), L762P (SEQ IDNO:161) and L763P (SEQ ID NO:159) were shown to be present in effusionfluid or sera of lung cancer patients but not in normal donors. Morespecifically, the presence of antibodies against L773PA, L514S, L523S,L762P and L763P in effusion fluid obtained from lung cancer patients andin sera from normal donors was detected by ELISA using recombinantproteins and HRP-conjugated anti-human Ig. Briefly, each protein (100ng) was coated in 96-well plate at pH 9.5. In parallel, BSA (bovineserum albumin) was also coated as a control protein. The signals ([S],absorbance measured at 405 nm) against BSA ([N]) were determined. Theresults of these studies are shown in Table 6, wherein − represents[S]/[N]<2; +/− represents [S]/[N]>2; ++ represents [S]/[N] >3; and +++represents [S]/[N]>5.

TABLE 6 Detection of Antibodies Against Lung Tumor Antigens L514S L523SL762P L763P L773PA Effusion fluid #1 +++ ++ ++ − ++ #2 − − +/− ++ +/− #3− − − − +/− #4 +/− ++ +/− − +/− #5 +/− +++ +/− +/− ++ #7 − +/− − − +/−#8 − +++ − − ++ #10 − ++ +/− +/− − #11 +/− ++ ++ − ++ #12 +++ +/− − +/−+/− #13 − +/− − − +/− #14 − +++ +/− +/− ++ #15 +/− ++ +/− − ++ #17 − +/−− − +/− #18 − ++ − − − #19 − +/− − − +/− #20 +/− +/− +/− − +/− Normalsera #21 − +/− − − − #22 − − − − − #23 − − − − +/− #24 − +/− − − − #25+/− +/− − − +/−

Using Western blot analyses, antibodies against L523S were found to bepresent in 3 out of 4 samples of effusion fluid from lung cancerpatients, with no L523S antibodies being detected in the three samplesof normal sera tested.

Example 17 EXPRESSION IN E. COLI OF A L514S HIS TAG FUSION PROTEIN

PCR was performed on the L514S-13160 coding region with the followingprimers:

(SEQ ID NO: 421) Forward primer PDM-278 5′ cacactagtgtccgcgtggcggcctac3′ Tm 67° C. (SEQ ID NO: 422) Reverse primer PDM-2805′ catgagaattcatcacatgcccttgaaggctccc 3′ TM 66° C.

The PCR conditions were as follows:

-   -   10 μl 10× Pfu buffer    -   1.0 μl 10 mM dNTPs    -   2.0 μl 10 μM each primer    -   83 μl sterile water    -   1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.)    -   50 ηg DNA

96° C. for 2 minutes, 96° C. for 20 seconds, 66° C. for 15 seconds, 72°C. for 1 minute with 40 cycles and then 72° C. for 4 minutes.

The PCR product was digested with EcoRI restriction enzyme, gel purifiedand then cloned into pPDM His, a modified pET28 vector with a His tag inframe, which had been digested with Eco72I and EcoRI restrictionenzymes. The correct construct was confirmed by DNA sequence analysisand then transformed into BL21 CodonPlus (Stratagene, La Jolla, Calif.)cells for expression.

The amino acid sequence of expressed recombinant L514S is shown in SEQID NO:423, and the DNA coding region sequence is shown in SEQ ID NO:424.

Example 18 EXPRESSION IN E. COLI OF A L523S HIS TAG FUSION PROTEIN

PCR was performed on the L523S coding region with the following primers:

(SEQ ID NO: 425) Forward primer PDM-4145′ aacaaactgtatatcggaaacctcagcgagaa 3′ Tm 62° C. (SEQ ID NO: 426)Reverse primer PDM-415 5′ ccatagaattcattacttccgtcttgactgagg 3′ TM 62° C.

The PCR conditions were as follows:

-   -   10 μl 10× Pfu buffer    -   1.0 μl 10 mM dNTPs    -   2.0 μl 10 μM each primer    -   83 μl sterile water    -   1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.)    -   50 ηg DNA

96° C. for 2 minutes, 96° C. for 20 seconds, 62° C. for 15 seconds, 72°C. for 4 minutes with 40 cycles and then 72° C. for 4 minutes.

The PCR product was digested with EcoRI restriction enzyme, gel purifiedand then cloned into pPDM His, a modified pET28 vector with a His tag inframe, which had been digested with Eco72I and EcoRI restrictionenzymes. The correct construct was confirmed by DNA sequence analysisand then transformed into BL21 CodonPlus (Stratagene, La Jolla, Calif.)cells for expression.

The amino acid sequence of expressed recombinant L523S is shown in SEQID NO:427, and the DNA coding region sequence is shown in SEQ ID NO:428.

Example 19 EXPRESSION IN E. COLI OF A L762PA HIS TAG FUSION PROTEIN

PCR was performed on the L762PA coding region (L762PA is missing thesignal sequence, the C-terminal transmembrane domain and the cytoplasmictail) with the following primers:

(SEQ ID NO: 355) Forward primer PDM-278 5′ggagtacagcttcaagacaatggg 3′ Tm57° C. (SEQ ID NO: 429) Reverse primer PDM-2795′ccatggaattcattatttcaatataagataatctc 3′ TM56° C.

The PCR conditions were as follows:

-   -   10 μl 10× Pfu buffer    -   1.0 μl 10 mM dNTPs    -   2.0 μl 10 μM each primer    -   83 μl sterile water    -   1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.)    -   50 ηg DNA

96° C. for 2 minutes, 96° C. for 20 seconds, 55° C. for 15 seconds, 72°C. for 5 minutes with 40 cycles and then 72° C. for 4 minutes.

The PCR product was digested with EcoRI restriction enzyme, gel purifiedand then cloned into pPDM His, a modified pET28 vector with a His tag inframe, which had been digested with Eco72I and EcoRI restrictionenzymes. The correct construct was confirmed by DNA sequence analysisand then transformed into BL21 pLys S (Novagen, Madison, Wis.) cells forexpression.

The amino acid sequence of expressed recombinant L762PA is shown in SEQID NO:430, and the DNA coding region sequence is shown in SEQ ID NO:431.

Example 20 EXPRESSION IN E. COLI OF A L773P HIS TAG FUSION PROTEIN

PCR was performed on the L773P coding region with the following primers:

(SEQ ID NO: 359) Forward primer PDM-299 5′ tggcagcccctcttcttcaagtggc 3′Tm 63° C. (SEQ ID NO: 432) Reverse primer PDM-3005′ cgcctgctcgagtcattaatattcatcagaaaatgg 3′ TM 63° C.

The PCR conditions were as follows:

-   -   10 μl 10× Pfu buffer    -   1.0 μl 10 mM dNTPs    -   2.0 μl 10 μM each primer    -   83 μl sterile water    -   1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.)    -   50 ηg DNA

96° C. for 2 minutes, 96° C. for 20 seconds, 63° C. for 15 seconds, 72°C. for 2 minutes 15 seconds with 40 cycles and then 72° C. for 4minutes.

The PCR product was digested with EcoRI restriction enzyme, gel purifiedand then cloned into pPDM His, a modified pET28 vector with a His tag inframe, which had been digested with Eco72I and EcoRI restrictionenzymes. The correct construct was confirmed by DNA sequence analysisand then transformed into BL21 pLys S (Novagen, Madison, Wis.) and BL21CodonPlus (Stratagene, La Jolla, Calif.) cells for expression.

The amino acid sequence of expressed recombinant L773P is shown in SEQID N0:433, and the DNA coding region sequence is shown in SEQ ID NO:434.

Example 21 CLONING AND SEQUENCING OF A T-CELL RECEPTOR CLONE FOR THELUNG SPECIFIC ANTIGEN L762P

T cell receptor (TCR) alpha and beta chains from a CD4 T cell clonespecific for the lung specific antigen L762P were cloned and sequence.Basically, total mRNA from 2×10⁶ cells from CTL clone 4H6 was isolatedusing Trizol reagent and cDNA was synthesized using Ready-to go kits(Pharmacia). To determine Valpha and Vbeta sequences of this clone, apanel of Valpha and Vbeta subtype specific primers was synthesized andused in RT-PCR reactions with cDNA generated from each of the clones.The RT-PCR reactions demonstrated that each of the clones expressed acommon Vbeta sequence that corresponded to the Vbeta8 subfamily and aValpha sequence that corresponded to the Valpha8 subfamily. To clone thefull TCR alpha and beta chains from clone 4H6, primers were designedthat spanned the initiator and terminator-coding TCR nucleotides. Theprimers were as follows:

forward primer for TCR Valpha8 5′ ggatccgccgccaccatgacatccattcgagctgta3′ (SEQ ID NO:435; has a BamHI site inserted);

Kozak reverse primer for TCR Valpha8 (antisense) 5′gtcgactcagctggaccacagccgcag 3′ (SEQ ID NO:436; has a SalI site insertedplus the TCR alpha constant sequence);

forward primer for TCR Vbeta8 (sense) 5′ggatccgccgccaccatggactcctggaccttctgct 3′ (SEQ ID NO:437; has a BamHIsite inserted); and

Kozak reverse primer for TCR Vbeta 5′ gtcgactcagaaatcctttctcttgac 3′

(SEQ ID NO:438; has a SalI site inserted plus the TCR beta constantsequence).

Standard 35 cycle RT-PCR reactions were established using the cDNAsynthesized from the CTL clone and the above primers utilizing theproofreading thermostable polymerase, PWO (Roche). The resultant PCRband, about 850 by for Valpha and about 950 for Vbeta, was ligated intoa PCR blunt vector (Invitrogen) and transformed into E. coli. E. colitransformed with plasmids having full-length alpha and beta chains wereidentified. Large scale preparations of the corresponding plasmids weregenerated, and these plasmids were sequenced.

The Valpha sequence (SEQ ID NO:439) was shown by nucleotide sequencealignment to be homologous to Valpha8.1, while the Vbeta sequence (SEQID NO:440) was shown by nucleotide sequence alignment to be homologousto Vbeta8.2.

Example 22 RECOMBINANT EXPRESSION OF FULL LENGTH L762P IN MAMMALIANCELLS

Full length L762P cDNA was subcloned into the mammalian expressionvectors VR1012 and pCEP4 (Invitrogen). Both expression vectors hadpreviously been modified to contain a FLAG epitope tag. These constructswere transfected into HEK293 and CHL-1 cells (ATCC) using Lipofectamine2000 reagent (Gibco). Briefly, both the HEK and CHL-1 cells were platedat a density of 100,000 cells/ml in DMEM (Gibco) containing 10% FBS(Hyclone) and grown overnight. The following day, 4 μl of Lipofectamine2000 was added to 100 μl of DMEM containing no FBS and incubated for 5minutes at room temperature. The Lipofectamine/DMEM mixture was thenadded to 1 μg of L762P Flag/pCEP4 or L762P Flag/VR1012 plasmid DNAresuspended in 100 μl DMEM and incubated for 15 minutes at roomtemperature. The Lipofectamine/DNA mix was then added to the HEK293 andCHL-1 cells and incubated for 48-72 hours at 37° C. with 7% CO₂. Cellswere rinsed with PBS, then collected and pelleted by centrifugation.L672P expression was detected in the transfected HEK293 and CHL-1 celllysates by Western blot analysis and was detected on the surface oftransfected HEK cells by flow cytometry analysis.

For Western blot analysis, whole cell lysates were generated byincubating the cells in Triton-X100 containing lysis buffer for 30minutes on ice. Lysates were then cleared by centrifugation at 10,000rpm for 5 minutes at 4° C. Samples were diluted with SDS-PAGE loadingbuffer containing beta-mercaptoethanol, then boiled for 10 minutes priorto loading the SDS-PAGE gel. The protein was transferred tonitrocellulose and probed using 1 μg/ml purified anti-L762P rabbitpolyclonal sera (lot #690/73) or non-diluted anti-L762P mAb 153.20.1supernatant. Blots were revealed using either goat anti-rabbit Igcoupled to HRP or goat anti-mouse Ig coupled to HRP followed byincubation in ECL substrate.

For flow cytometric analysis, cells were washed further with ice coldstaining buffer (PBS+1% BSA+Azide). Next, the cells were incubated for30 minutes on ice with 10 ug/ml of purified anti-L762P polyclonal sera(lot #690/73) or a 1:2 dilution of anti-L762P mAb 153.20.1 supernatant.The cells were washed 3 times with staining buffer and then incubatedwith a 1:100 dilution of goat anti-rabbit Ig(H+L)-FITC or goatanti-mouse Ig(H+L)-FITC reagent (Southern Biotechnology) for 30 minuteson ice. After 3 washes, the cells were resuspended in staining buffercontaining propidium iodide (PI), a vital stain that allows for theexclusion of permeable cells, and analyzed by flow cytometry.

Example 23 GENERATION OF POLYCLONAL ANTIBODIES TO LUNG TUMOR ANTIGENS

Three lung antigens, L523S (SEQ ID NO:176), L763P (SEQ ID NO:159) andL763 peptide #2684 (SEQ ID NO:441), were expressed and purified for usein antibody generation.

L523S and L763P were expressed in an E. coli recombinant expressionsystem and grown overnight in LB Broth with the appropriate antibioticsat 37° C. in a shaking incubator. The next morning, 10 ml of theovernight culture was added to 500 ml of 2× YT with the appropriateantibiotics in a 2L-baffled Erlenmeyer flask. When the optical densityof the culture reached 0.4-0.6 at 560 nanometers, the cells were inducedwith IPTG (1 mM). Four hours after induction with IPTG, the cells wereharvested by centrifugation.

The cells were then washed with phosphate buffered saline andcentrifuged again. The supernatant was discarded and the cells wereeither frozen for future use or immediately processed. Twentymilliliters of lysis buffer was added to the cell pellets and vortexed.To break open the E. coli cells, this mixture was then run through afrench press at a pressure of 16,000 psi. The cells were thencentrifuged again and the supernatant and pellet were checked bySDS-PAGE for the partitioning of the recombinant protein.

For proteins that localized to the cell pellet, the pellet wasresuspended in 10 mM Tris pH 8.0, 1% CHAPS and the inclusion body pelletwas washed and centrifuged again. This procedure was repeated twicemore. The washed inclusion body pellet was solubilized with either 8Murea or 6M guanidine HCl containing 10 mM Tris pH 8.0 plus 10 mMimidazole. The solubilized protein was added to 5 ml of nickel-chelateresin (Qiagen) and incubated for 45 minutes to 1 hour at roomtemperature with continuous agitation.

After incubation, the resin and protein mixture was poured through adisposable column and the flow through was collected. The column wasthen washed with 10-20 column volumes of the solubilization buffer. Theantigen was then eluted from the column using 8M urea, 10 mM Tris pH 8.0and 300 mM imidazole and collected in 3 ml fractions. A SDS-PAGE gel wasrun to determine which fractions to pool for further purification.

As a final purification step, a strong anion exchange resin, in thiscase Hi-Prep Q (Biorad), was equilibrated with the appropriate bufferand the pooled fractions from above were loaded onto the column. Eachantigen was eluted off the column with an increasing salt gradient.Fractions were collected as the column was run and another SDS-PAGE gelwas run to determine which fractions from the column to pool.

The pooled fractions were dialyzed against 10 mM Tris pH 8.0. Therelease criteria were purity as determined by SDS-PAGE or HPLC,concentration as determined by Lowry assay or Amino Acid Analysis,identity as determined by amino terminal protein sequence, and endotoxinlevel was determined by the Limulus (LAL) assay. The proteins were thenput in vials after filtration through a 0.22-micron filter and theantigens were frozen until needed for immunization.

The L763 peptide #2684 was synthesized and conjugated to KLH and frozeuntil needed for immunization.

The polyclonal antisera were generated using 400 micrograms of each lungantigen combined with 100 micrograms of muramyldipeptide (MDP). An equalvolume of Incomplete Freund's Adjuvant (IFA) was added and then mixedand injected subcutaneously (S.C.) into a rabbit. After four weeks, therabbit was S.C. boosted with 200 micrograms of antigen mixed with anequal volume of IFA. Thereafter the rabbit was I.V. boosted with 100micrograms of antigen. The animal was bled seven days following eachboost. The blood was then incubated at 4° C. for 12-24 hours followed bycentrifugation to generate the sera.

The polyclonal antisera were characterized using 96 well plates coatedwith antigen and incubated with 50 microliters (typically 1microgram/microliter) of the polyclonal antisera at 4° C. for 20 hours.Basically, 250 microliters of BSA blocking buffer was added to the wellsand incubated at room temperature for 2 hours. Plates were washed 6times with PBS/0.1% Tween. The rabbit sera were diluted in PBS/0.1%Tween/0.1% BSA. 50 microliters of diluted sera was added to each welland incubated at room temperature for 30 minutes. The plates were washedas described above, and then 50 microliters of goat anti-rabbithorseradish peroxidase (HRP) at a 1:10000 dilution was added andincubated at room temperature for 30 minutes.

The plates were washed as described above, and 100 microliters of TMBMicrowell Peroxidase Substrate was added to each well. Following a15-minute incubation in the dark at room temperature, the colorimetricreaction was stopped with 100 microliters of 1N H₂SO₄ and readimmediately at 450 nm. All the polyclonal antibodies showedimmunoreactivity to the appropriate antigen. Tables 7-9 show theantibody reactivity of rabbit antisera in serial dilution to the threelung antigens, L523S, L763P and L763 peptide #2684. The first columnshows the antibody dilutions. The columns “Pre-immune sera” indicateELISA data for two experiments using pre-immune sera. These results areaveraged in the fourth column. The columns “anti-L523S, L763P or #2684”indicate ELISA data for two experiments using sera from rabbitsimmunized as described in this Example, using the respective antigen,referred to as either L523S, L763P or #2684 in the tables.

TABLE 7 Pre- immune Pre- Anti- Anti- Antibody sera immune L523S L523Sdilution (1) sera (2) Average (1) (2) Average 1:1000 0.14 0.14 0.14 2.362.37 2.37 1:2000 0.12 0.10 0.11 2.29 2.23 2.26 1:4000 0.10 0.09 0.102.11 2.17 2.14 1:8000 0.09 0.09 0.09 1.98 2.00 1.99 1:16000 0.09 0.090.09 1.73 1.76 1.75 1:32000 0.09 0.09 0.09 1.35 1.40 1.37 1:64000 0.090.11 0.10 0.94 0.98 0.96 1:128000 0.09 0.08 0.08 0.61 0.61 0.61 1:2560000.08 0.08 0.08 0.38 0.38 0.38 1:512000 0.09 0.08 0.08 0.24 0.25 0.251:1024000 0.08 0.08 0.08 0.17 0.17 0.17 1:2048000 0.08 0.08 0.08 0.140.13 0.13

TABLE 8 Pre- immune Pre- Anti- Anti- Antibody sera immune L763P L763Pdilution (1) sera (2) Average (1) (2) Average 1:1000 0.09 0.11 0.10 1.971.90 1.93 1:2000 0.07 0.07 0.07 1.86 1.84 1.85 1:4000 0.06 0.06 0.061.82 1.81 1.81 1:8000 0.06 0.06 0.06 1.83 1.81 1.82 1:16000 0.06 0.050.06 1.79 1.74 1.76 1:32000 0.06 0.06 0.06 1.56 1.51 1.53 1:64000 0.060.05 0.05 1.35 1.34 1.35 1:128000 0.05 0.05 0.05 1.01 0.98 0.99 1:2560000.06 0.05 0.05 0.69 0.70 0.70 1:512000 0.06 0.05 0.05 0.47 0.44 0.461:1024000 0.06 0.05 0.06 0.27 0.27 0.27 1:2048000 0.05 0.05 0.05 0.160.15 0.16

TABLE 9 Pre- immune Pre- Anti- Anti- Antibody sera immune #2684 #2684dilution (1) sera (2) Average (1) (2) Average 1:1000 0.07 0.07 0.07 2.102.00 2.05 1:2000 0.07 0.06 0.06 1.95 1.96 1.95 1:4000 0.06 0.06 0.061.77 1.82 1.79 1:8000 0.06 0.06 0.06 1.79 1.81 1.80 1:16000 0.06 0.060.06 1.54 1.50 1.52 1:32000 0.06 0.06 0.06 1.27 1.20 1.24 1:64000 0.060.06 0.06 0.85 0.82 0.83 0 0.06 0.06 0.06 0.06 0.06 0.06

Tables 10-12 show the affinity purification of the respective antibodiesto the three lung antigens, L523S, L763P and L763 peptide #2684.

TABLE 10 Affinity Affinity Affinity Affinity Antibody pure pure purepure conc. (salt (salt (acid (acid (μg/ml) peak) peak) Average peak)peak) Average 1.0 2.38 2.35 2.36 2.25 2.31 2.28 0.5 2.24 2.22 2.23 2.192.18 2.18 0.25 2.05 2.09 2.07 2.01 2.03 2.02 0.13 1.70 1.81 1.75 1.741.74 1.74 0.063 1.44 1.44 1.44 1.43 1.38 1.40 0.031 1.05 1.05 1.05 0.990.99 0.99 0.016 0.68 0.67 0.68 0.65 0.64 0.64 0.0078 0.43 0.42 0.42 0.390.39 0.39 0.0039 0.27 0.26 0.27 0.24 0.26 0.25 0.0020 0.18 0.20 0.190.19 0.18 0.19 0.0010 0.13 0.14 0.13 0.13 0.14 0.13 0.00 0.11 0.12 0.110.10 0.12 0.11

TABLE 11 Antibody Affinity Affinity dilution pure pure Average 1:10001.64 1.77 1.70 1:2000 1.59 1.76 1.68 1:4000 1.48 1.62 1.55 1:8000 1.351.43 1.39 1:16000 1.09 1.19 1.14 1:32000 0.81 0.89 0.85 1:64000 0.550.58 0.56 1:128000 0.31 0.35 0.33 1:256000 0.18 0.20 0.19 1:512000 0.110.12 0.11 1:1024000 0.07 0.07 0.07 1:2048000 0.06 0.06 0.06

TABLE 12 Antibody conc. Affinity Affinity (μg/ml) pure pure Average 1.02.00 2.02 2.01 0.5 2.01 1.93 1.97 0.25 1.84 1.83 1.84 0.13 1.80 1.831.81 0.06 1.39 1.60 1.50 0.03 1.33 1.35 1.34 0.02 0.94 0.93 0.94 0.000.06 0.06 0.06

Example 24 FULL-LENGTH CDNA SEQUENCE ENCODING L529S

The isolation of a partial sequence (SEQ ID NO:106) for lung antigenL529S was previously provided in Example 2. This partial sequence wasused as a query to identify potential full length cDNA and proteinsequences by searching against publicly available databases. Thepredicted full-length cDNA sequence for the isolated cloned sequence ofSEQ ID NO:106 is provided in SEQ ID NO:442. The deduced amino acidsequence of the antigen encoded by SEQ ID NO:442 is provided in SEQ IDNO:443. It was previously disclosed in Example 2 that L529S showssimilarity to connexin 26, a gap junction protein.

Example 25 EXPRESSION IN MEGATERIUM OF A HISTIDINE TAG-FREE L523S FUSIONPROTEIN

PCR was performed on the L523S coding region with the following primers:

(SEQ ID NO: 444) Forward primer PDM-7345′ caatcaggcatgcacaacaaactgtatatcggaaac 3′ Tm 63° C. (SEQ ID NO: 445)Reverse primer PDM-735 5′ cgtcaagatcttcattacttccgtcttgac 3′ TM 60° C.

The PCR conditions were as follows:

-   -   10 μl 10× Pfu buffer    -   1.0 μl 10 mM dNTPs    -   2.0 μl 10 μM each primer    -   83 μl sterile water    -   1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.)    -   50 ηg DNA

96° C. for 2 minutes, 96° C. for 20 seconds, 62° C. for 15 seconds, 72°C. for 4 minute with 40 cycles and then 72° C. for 4 minutes.

The PCR product was digested with SphI and BgIII restriction enzymes,gel purified and then cloned into pMEG-3, which had been digested withSphI and BgIII restriction enzymes. The correct construct was confirmedby DNA sequence analysis and then transformed into Megaterium cells forexpression.

The amino acid sequence of expressed recombinant L523S is shown in SEQID NO:446, and the DNA coding region sequence is shown in SEQ ID NO:447.

Example 26 EXPRESSION IN E. COLI OF A HISTIDINE TAG-FREE L523S FUSIONPROTEIN

PCR was performed on the L523S coding region with the following primers:

(SEQ ID NO: 448) Forward primer PDM-7335′ cgtactagcatatgaacaaactgtatatcggaaac 3′ Tm 64° C. (SEQ ID NO: 426)Reverse primer PDM-415 5′ ccatagaattcattacttccgtcttgactgagg 3′ TM 62° C.

The PCR conditions were as follows:

-   -   10 μl 10× Pfu buffer    -   1.0 μl 10 mM dNTPs    -   2.0 μl 10 μM each primer    -   83 μl sterile water    -   1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, Calif.)    -   50 ηg DNA

96° C. for 2 minutes, 96° C. for 20 seconds, 62° C. for 15 seconds, 72°C. for 4 minute with 40 cycles and then 72° C. for 4 minutes.

The PCR product was digested with NdeI and EcoRI restriction enzymes,gel purified and then cloned into pPDM, a modified pET28 vector, whichhad been digested with NdeI and EcoRI restriction enzymes. The correctconstruct was confirmed by DNA sequence analysis and then transformedinto BLR pLys S and HMS 174 pLys S cells for expression.

The amino acid sequence of expressed recombinant L523S is shown in SEQID NO:449, and the DNA coding region sequence is shown in SEQ ID NO:450.

Example 27 EPITOPE-ANALYSIS OF L514S AND L523S-SPECIFIC ANTIBODIES

Peptides of candidate antigens can be used for the evaluation ofantibody responses in both preclinical and clinical studies. These dataallow one to further confirm the antibody response against a certaincandidate antigen.

Protein-based ELISA with and without competitive peptides andpeptide-based ELISA can be used to evaluate these antibody responses.Peptide ELISA is especially useful since it can further exclude thefalse positive of the antibody titer observed in protein-based ELISA aswell as to provide the simplest assay system to test antibody responsesto candidate antigens. In this example, data was obtained using bothL514S- and L523S-peptides that show that individual cancer patientsproduce L514S- and L523S-specific antibodies. The L514S-specificantibodies recognize primarily the following epitope of L514S:

(SEQ ID NO: 451) aa86-110 LGKEVRDAKITPEAFEKLGFPAAKE.

This epitope is the common epitope in humans. A rabbit antibody specificfor L514S recognizes two addition epitopes of L514S:

(SEQ ID NO: 452) (1) aa21-45: KASDGDYYTLAVPMGDVPMDGISVA (SEQ ID NO: 453)(2) aa121-135: PDRDVNLTHQLNPKVK

It was further found that the SEQ ID NO:452 is common to both L514Sisoforms, L514S-13160 and L514S-13166, whereas the other epitopes, SEQID NO:451 and SEQ ID NO:453, are probably specific to the isoform,L514S-13160.

The L523S-specific antibodies recognize primarily the following epitopeof L523S:

aa440-460: KIAPAEAPDAKVRMVIITGP. (SEQ ID NO: 454)

This epitope is the common epitope in humans. A rabbit antibody specificfor L523S recognizes two other epitopes:

(SEQ ID NO: 455) (1) aa156-175 PDGAAQQNNNPLQQPRG (SEQ ID NO: 456) (2)aa326-345: RTITVKGNVETCAKAEEEIM

In further studies, it was determined by peptide based ELISAs that eightadditional epitopes of L523S were recognized by L523S-specificantibodies:

(SEQ ID NO: 457) (1) aa40-59 AFVDCPDESWALKAIEALS (SEQ ID NO: 458) (2)aa80-99: IRKLQIRNIPPHLQWEVLDS (SEQ ID NO: 459) (3) aa160-179:AQQNPLQQPRGRRGLGQRGS (SEQ ID NO: 460) (4) aa180-199:DVHRKENAGAAEKSITILST (SEQ ID NO: 461) (5) aa320-339:LYNPERTITVKGNVETCAKA (SEQ ID NO: 462) (6) aa340-359:EEEIMKKIRESYENDIASMN (SEQ ID NO: 463) (7) aa370-389:LNALGLFPPTSGMPPPTSGP (SEQ ID NO: 464) (8) aa380-399:KIAPAEAPDAKVRMVIITGP

Out of these, six epitopes are common in both lung pleural effusionfluid samples and in sera of lung patients. Of these six, SEQ ID NO:459and SEQ ID NO:463 have no homology to other L523S-family proteins suchas IGF-II mRNA-binding proteins 1 and 2. Accordingly, this indicatesthat these two peptides can be used as an assay system to determine theantibody response to L523S.

Example 28 GENERATION OF L523S-SPECIFIC CTL LINES USING IN VITROWHOLE-GENE PRIMING

To determine if L523S is capable of generating a CD8⁺ T cell immuneresponse, CTLs were generated using in vitro whole-gene primingmethodologies with tumor antigen-vaccinia infected DC (Yee et al, TheJournal of Immunology, 157(9):4079-86, 1996), human CTL lines werederived that specifically recognize autologous fibroblasts transducedwith the L523S tumor antigen, as determined by interferon-gamma ELISPOTanalysis. Specifically, dendritic cells (DC) were differentiated fromPercoll-purified monocytes derived from PBMC of normal human donors byplastic adherence and growing for five days in RPMI medium containing10% human serum, 50 ng/ml human GM-CSF and 30 ng/ml human IL-4.Following the five days of culture, the DC were infected overnight witha recombinant adenovirus that expresses L523S at a multiplicity ofinfection (M.O.I) of 33, 66 and 100, and matured overnight by theaddition of 2 μg/ml CD40 ligand. The virus was then inactivated by gammairradiation. In order to generate a CTL line, autologous PBMC wereisolated and CD8+ T cells were enriched for by the negative selectionusing magnetic beads conjugated to CD4+, CD14+, CD16+, CD19+, CD34+ andCD56+ cells. CD8+ T cells specific for L523S were established in roundbottom 96-well plates using 10,000 L523S expressing DCs and 100,000 CD8+T cells per well in RPMI supplemented with 10% human serum, 10 ng/ml ofIL-6 and 5 ng/ml of IL-12. The cultures were restimulated every 7-10days using autologous primary fibroblasts retrovirally transduced withL523S, and the costimulatory molecule CD80 in the presence of IL-2. Thecells were also stimulated with IFN-gamma to upregulate MHC Class I. Themedia was supplemented with 10 U/m1 of IL-2 at the time of stimulationas well as on days 2 and 5 following stimulation. Following threestimulation cycles, ten L523S specific CD8+ T cell lines were identifiedusing interferon-gamma ELISPOT analysis that specifically produceinterferon-gamma when stimulated with the L523S tumor antigen-transducedautologous fibroblasts, but not with a control antigen.

One line, 6B1, was cloned using anti-CD3 and feeder cells. The cloneswere tested for specificity on L523S-transduced fibroblasts. Inaddition, using a panel of HLA-mismatched lines transduced with a vectorexpressing L523S and measuring interferon-gamma production by this CTLline in an ELISPOT assay, it was determined that this clone 6B1.4B8 isrestricted by HLA-A0201.

Also using transfected Cos cells, it was shown that clone 6B1.4B8recognizes Cos cells transfected with pcDNA3 HLA A0201/L523S in anHLA-restricted and antigen specific manner.

An epitope mapping study demonstrated the clone 6B1.4B8 recognizesHLA-A201 LCL loaded with peptide pool 3 (a polypeptide corresponding toamino acid positions 33-59 of L523S.

A peptide pool breakdown study demonstrated that clone 6B1.4B8recognizes autologous B-LCL loaded with 15-mer peptides from amino acidpositions 37-55 of L523S, TGYAFVCPDESWALKAIE (SEQ ID NO:465). A furtherpeptide breakdown study demonstrated that clone 6B1.4B8 recognizes T2cells loaded with the same 15-mer peptides.

A peptide recognition study demonstrated that clone 6B1.4B8 prefers T2cells loaded with the peptide FVDCPESWAL (SEQ ID NO:466) which iscorresponds to the amino acid sequence at positions 41-51 of L523S andis encoded by the DNA sequence of SEQ ID NO:467.

Example 29 L523S EXPRESSION IN OTHER HUMAN CANCERS

It was previously disclosed in Example 2 that L523S is expressed in lungcancers including squamous, adenocarcinoma and small cell carcinoma. Tofurther evaluate the expression profile of this antigen an electronicexpress profiling was performed. This was done by searching aL523S-specific sequence against a public EST database. Results of thisprofiling indicate that L523S may also be present in colonadenocarcinomas, prostate adenocarcinomas, CML, AML, Burkitt's Lymphoma,brain tumors, retinoblastomas, ovarian tumors, teratocarcinomas, uterusmyosarcomas, germ cell tumors as well as pancreatic and cervical tumorcell lines.

Example 30 IMMUNOHISTOCHEMISTRY ANALYSIS OF L523S

In order to determine which tissues express the lung tumor antigenL523S, immunohistochemistry (IHC) analysis was performed on a diverserange of tissue types. Polyclonal antibodies specific for L523S (SEQ IDNO:176) were generated as described in Example 23. IHC was performedessentially as described in Example 6. Briefly, tissue samples werefixed in formalin solution for 12-24 hours and embedded in paraffinbefore being sliced into 8 micron sections. Steam heat induced epitoperetrieval (SHIER) in 0.1 sodium citrate buffer (pH 6.0) was used foroptimal staining conditions. Sections were incubated with 10% serum inPBS for 5 minutes. The primary L523S antibody was added to each sectionfor 25 minutes followed by a 25 minute incubation with anti-rabbitbiotinylated antibody. Endogenous peroxidase activity was blocked bythree 1.5 minute incubations with hydrogen peroxidase. The avidin biotincomplex/horse radish peroxidase (ABC/HRP) system was used along with DABchromogen to visualize antigen expression. Slides were counterstainedwith hematoxylin to visualize the cell nuclei.

IHC analysis of L523S expression revealed that of the lung cancertissues tested over 90% of tissue samples demonstrated highover-expression of the lung tumor antigen (10/11 adenocaricomas and 8/9squamous). Of the normal tissues tested, all were negative forexpression of L523S, with the exception of weak staining in normalbronchus, testis, liver, and trachea.

Example 31 GENERATION AND CHARACTERIZATION OF L762 HUMAN MONOCLONALANTIBODIES

Cell supernatants from hybridoma fusions from the Xenomouse strain oftransgenic mice were screened for ability to bind to L762P. All resultsare shown in Table 13. The primary screen was to test monoclonalsupernatants for reactivity to L762P by ELISA analysis using recombinantbacterial expressed protein. We next tested the human supernatants forreactivity to surface expressed L762P by whole cell ELISA usingfluorimetry analysis. Specific reactivity of the humab supernatants wasconfirmed by performing FACS analysis on cells transfected with eitheran irrelevant plasmid or a plasmid expressing L762P. FI/CFI is therelative fold increase in fluorescence intensity (FI) of the anti-L762Phumab primary antibody to irrelevant human primary antibody. FI/CFI/A20is the relative fold increase in fluorescence intensity (FI) of theanti-L762P humab primary antibody to irrelevant human primary antibodyover the FI of the anti-L762P mouse monoclonal antibody 153A20.1.FI/CFI/R690 is the relative fold increase in fluorescence intensity (FI)of the anti-L762P humab primary antibody to irrelevant human primaryantibody over the FI of the anti-L762P rabbit polyclonal antibody. FACSVRL762 is the percentage of cells transfected with plasmid expressingL762P that were positive following staining with indicated monoclonalantibody. FACS VR(−) is the percentage of cells transfected withirrelevant plasmid that were positive following staining with indicatedmonoclonal antibody. ELISA is the O.D. values of the indicatedmonoclonal antibody to recombinant L762P protein. The shaded rows inTable 13 indicate those antibodies that will be further cloned andcharacterized.

For generation of mouse monoclonal antibodies 153Al2.1 and 153A20.1,Balb/c mice were immunized with E. coli recombinant L762P protein (aminoacid residues 32-944 of SEQ ID NO:161). The mice were subsequently usedfor splenic B cell fusions to generate anti-L762P hybridomas. Twoclones: 153A12.1 and 153A20.1 (IgG2a, kappa) were grown for antibodyproduction and the secreted monoclonal antibody was purified by passingspent culture supernatants over a Protein A-Sepharose column, followedby antibody elution using 0.2M glycine, pH 2.3. Purified antibody wasneutralized by the addition of 1M Tris, pH 8 and buffer exchanged intoPBS.

TABLE 13 Human Monoclonal Antibodies Against L762P

for 1.170 to 1.175 FI-fluorescence intensity of primary antibodyCFI-fluorescence intensity of human irrelevant primary antibody.A20-mouse anti-L762P monoclonal antibody R690-rabbit anti-L762P affinitypurified polyclonal antibody FACS VRL762-percent positive cells fromtransient transfection of VR1013/L762 expression plasmid FACSVR(−)-percent positive cells from transient transfection of empty VR1013expression plasmid

Example 32 EPITOPE MAPPING AND PURIFICATION OF HL523S-SPECIFICANTIBODIES

This Example describes the purification of L523S antibodies that candistinguish between human and mouse L523S homologs and will likelydistinguish between hL523S and hL523S-family members such as hIMP-1 andhIMP-2.

L523S (full-length cDNA and amino acid sequence set forth in SEQ IDNO:347 and 348, respectively) is one of a family of proteins thatincludes hIMP-1 and hIMP-2. The members of this family of proteins havea high degree of similarity one to the other and are also highly similarbetween species. Thus, generating antibodies that specifically recognizehuman L523S (hL523S) and not other members of the protein family inhumans or the mouse homologs, has been problematic. However, in order toevaluate preclinical and clinical L523S DNA/Adenoviral vaccines bydetecting the protein expression of L523S, human L523S-specificantibodies are critical.

Polyclonal antibodies specific for hL523S were generated as described inExample 23. These antibodies were used to map epitopes. The epitopeanalysis showed 2 particular peptides of hL523S that were recognized,peptide 16/17 and peptide 32.

The amino acid sequences of both hL523S and mouse L523S (mL523S) peptide16/17 and peptide 32 were then compared. Peptide 32/33 is identicalbetween hL523S and mL523S. However, as the alignment below indicates,peptide 16/17 has 5 amino acid differences between the human and mousehomologs (underlined).

hL523S (16/17) (SEQ ID NO: 468): IPDEMAAQQNPLQQPRGRRGLGQR mL523S (16/17)(SEQ ID NO: 469): IPDETAAQQNPSPQLRGRRGPGQR

Moreover, peptide-based ELISAs showed that peptide 17 is specificallyrecognized by lung cancer patient sera #197, and a homology search ofpeptide 17 between human IMP (hIMP) family members shows that there islittle similarity in this region between family members. The hL523Speptide 17 (and 16/17) has less than 50% similarity to hL523S familymembers such as hIMP-1 and hIMP-2.

Based upon the epitope mapping of L523S-specific antibodies and the datafrom the homology search, hL523S or mL523S peptide 16/17-conjugatedligands were then used to purify human or mouse L523S-specificantibodies from rabbit polyclonal antibodies generated against hL523Sprotein as described in Example 23. The data from the antibodiespurified by affinity chromatography using ligands conjugated with eitherhL523S-peptide 16/17 or mL523S-peptide 16/17 suggested that the affinityof antibodies specific to hL523S-peptide 16/17 is much higher than thatof antibodies to mL523S-peptide 16/17 since they bind more strongly tohL523S-peptide 16/17 than to mL523S-peptide 16/17. The difference inaffinity between the purified antibodies to human and mouseL523S-peptide 16/17 was confirmed by peptide-based ELISA. The antibodiespurified by hL523S-peptide 16/17 selectively bind to human L523S-peptide16/17 but bind much less or not at all to mL523S-peptide 16/17.

In order to further characterize the original polyclonal antibodies andantibodies purified by hL523S-peptide 16/17, immunoblot analysis wasconducted using both human lung adenocarcinoma line as a source ofhL523S protein and mouse whole body embryo (day 17 gestation) as thesource of mL523S protein. This analysis showed that polyclonalantibodies specific for hL523S recognize hL523S protein expressed in thetumor cell line as well as mL523S protein expressed in whole bodyembryos of day 17 gestation. However, the addition of hL523S peptide32/33 blocks binding of antibodies to human and mouse L523S proteins.Thus, the crossreactivity of the polyclonal antibodies to mL523S proteinis due to the existence of antibodies specific to hL523S peptide 32/33.In marked contrast, the purified antibodies specific to hL523S peptide16/17 do not bind mL523S protein expressed in mice embryos but dorecognize hL523S protein expressed in human lung adenocarcinoma cells.These data confirm the ELISA data using hL523S-peptide 16/17 andmL523S-peptide 16/17 described above.

The amino acid sequence of hL523S peptide 16/17 used to purify theantibodies is about 60-70% similar to that of the mL523S-peptide 16/17which is not recognized by hL523S-specific antibodies by Western blotanalysis and peptide-based ELISA. The hL523S peptide 16/17 has less than50% similarity to hL523S family members such as hIMP-1 and hIMP-2. Takentogether, these data suggest that it is highly probable that theantibodies purified by hL523S peptide 16/17 described herein will alsodistinguish hL523S protein from the other hL523S family members.

In summary, antibodies purified with the hL523S peptide 16/17 do notrecognize the mouse L523S homolog. The amino acid sequence of peptide16/17 between hL523S family members is less similar than between humanand mouse L523S. Thus, the hL523S-specific antibodies described abovecan be used to distinguish between human and mouse L523S and betweenmembers of the hL523S family of proteins and can therefore be used forthe accurate detection of hL523S protein expression in animals andhumans.

Example 33 IN VIVO IMMUNOGENECITY OF LUNG TUMOR ANTIGEN L523

This example describes two in vivo immunogenicity studies to evaluatethe vaccination of mice with either an adenovirus containing L523 orwith L523 naked DNA followed by a second immunization with an adenoviruscontaining L523.

The first study involved the immunization of two strains of mice withL523 adenovirus. The C57Bl6 strain of mice is homozygous for HLA-typeH-2^(b), while strain B6D2(F1) is heterozygous for the HLA-type,H-2^(b/d). Table 14 describes the initial immunization strategyemployed.

TABLE 14 Immunization with L523 Adenovirus alone: Experimental DesignGroup Immunization Strain (4/group) 1 10⁸ PFU Ad L523 A C57BL6 2 10⁷ PFUAd hrGFP A C57BL6 3 10⁸ PFU Ad L523 A B6D2(F1) 4 10⁷ PFU Ad hrGFP AB6D2(F1) 5 Naïve C57BL6 6 Naïve B6D2(F1) PFU = plaque forming unit; GFP= green fluorescent protein; Ad = adenovirus.

Mice were immunized intradermally with either 10⁸ PFU of L523-adenovirusor 10⁷ PFU of an irrelevant adenovirus (hrGFP). Three weeks followingimmunization, IgG1 and IgG2a antibody responses to L523 were examined inall groups of mice. Briefly, recombinant full length L523 (rL523) wascoated onto ELISA plates and serum, at multiple dilutions, was added tothe wells. Following a 60-minute incubation, the serum was washed fromthe wells and a secondary antibody, either specific for an IgG1 or IgG2awas added to the plates. Both antibodies were directly conjugated tohorseradish peroxide (HRP). The levels of L523 antibodies, either IgG1or IgG2a, were measured in all groups. In the C57BL6 mice, little to noL523-specific antibodies were detected following immunization. However,in the B6D2(F1) strain of mice immunized with L523 adenovirus, both IgG1and IgG2a L523-specific antibodies were detected at serum dilution aslow as 1/1000.

In addition to detecting L523-specific antibodies in the serum,interferon-gamma (IFN-γ) responses were assayed from immune spleen cellsfollowing in vitro stimulation with rL523 protein. Briefly, spleen cellswere harvested from all mice groups and cultured for 3 days in 96-wellplates. Culture conditions included, media alone, 1 or 10 μg/ml of rL523protein, or 5 μg/ml of concanavalin A (Con A). After 3 days, thesupernatants were harvested and assayed for IFN-γ levels in thesupernatants.

Immunization with L523-adenovirus, but not an irrelevant adenovirus,elicited a strong IFN-γ response from the spleen cells which werestimulated with rL523. In general, responses were stronger in theB6D2(F1) mouse strain, as evidenced by both a higher level of IFN-γproduction, as well as the fact that stimulation with a lower antigenconcentration (1 μg/ml) elicited an equally strong response as seen withthe higher antigen concentration (10 μg/ml).

Finally, T cell proliferation responses were assayed from immune spleencells by stimulation in vitro with rL523 protein. Briefly, spleen cellswere cultured for 4 days in 96-well plates with, media alone, 1 or 10μg/ml of rL523 protein, or Con A. The cultures were then pulsed with3H-thymidine for the final 8 hours of culture. Results are representedas the stimulation index (SI) in the presence of antigen relative tostimulation with media alone. Results were consistent with thoseobtained in the IFN-γ assay. Immunization with L523-adenovirus, but notan irrelevant adenovirus, elicited a proliferation response in spleencells stimulated with rL523. A strong SI (average of >20) was observedin spleen cells harvested from the B6D2(F1) mouse strain, with similarlevels of proliferation observed at both protein concentrations. Littleor no T cell proliferation was observed in the C57BL6 mouse strain.

A second study involved the immunization of two strains of miceinitially with L523 naked DNA followed by a second immunization withL523 adenovirus two weeks later. The mice were harvested 3 weeks afterthe boost. Table 15 describes the immunization regimen of the secondstudy.

TABLE 15 Immunization with L523 DNA followed by a second immunizationwith L523-Adenovirus: Experimental Design Group Immunization Strain(4/group) 1 L523 DNA + 10⁸ PFU Ad L523 A C57BL6 2 10⁸ PFU Ad L523 AC57BL6 3 Irrelevant DNA + 10⁷ PFU Ad hrGFP A C57BL6 4 10⁷ PFU Ad hrGFP AC57BL6 5 Naive C57BL6 6 L523 DNA + 10⁸ PFU Ad L523 A B6D2(F1) 7 10⁸ PFUAd L523 A B6D2(F1) 8 Irrelevant DNA + 10⁷ PFU Ad hrGFP A B6D2(F1) 9 10⁷PFU Ad hrGFP A B6D2(F1) 10 Naïve B6D2(F1) PFU = plaque forming unit; GFP= green fluorescent protein; Ad = adenovirus.

As described in the first study, strong IgG1 and IgG2a antibodyresponses were observed in B6D2(F1) mice following immunization withL523-adenovirus. Immunizing with L523 DNA appeared to increase theoverall L523-specific antibody response compared to responses achievedwith immunization with L523-adenovirus alone. C57BL6 mice elicitedlittle or no L523-specific antibody responses following immunizationwith L523-adenovirus, but were some slightly positive responses weredetected in mice immunized with L523 DNA followed by a secondimmunization with L523-adenovirus.

IFN-γ responses were assayed from immune spleen cells by stimulation invitro with rL523 protein. These results confirm those observed in theinitial study demonstrating the immunogenecity of L523 in animals. Theresults also suggest that initially immunizing the animals with L523DNA, prior to immunization with L523-adeonvirus, does not significantlyincrease the CD4 response. As with the initial study, responses appearto be stronger in the B6D2(F1) strain of mice than the C57BL6 strain.

As with the initial study, T cell proliferation responses were assayedfrom immune spleen cells by stimulation in vitro with rL523 protein. Theresults from using two rounds of immunization are consistent with thoseobtained from the first study. Immunization with L523 DNA prior to asecond round of immunization with L523-adenovirus did not significantlyincrease the proliferation responses generated in the mice. As with thefirst study, responses were stronger in the B6D2(F1) mouse strain thanin the C57BL6 strain.

The difference in HLA types between the two strains of mice couldexplain variations in the extent of the immune responses detected. Asdescribed above, the C57BL6 strain is homozygous for H-2^(b), while theB6D2(F1) is heterozygous for H-2^(b/d). The increased diversity of theB6D2(F1) strains HLA type allows for a greater number of epitopesderived from the L523 protein to be presented. In this strain, epitopesspecific for both H-2^(b) and H-2^(d) can be presented, while onlyH-2^(b) epitopes can be presented by the C57BL6 strain.

Example 34 GENERATION OF MOUSE MONOCLONAL ANTIBODIES TO L523SRECOMBINANT PROTEIN

This example describes the generation of mouse monoclonal antibodiesspecific for the lung tumor antigen, L523S. These data show that L523Sis immunogenic and support its use to generate B cell immune responsesin vivo. Further, the antibodies generated herein can be used indiagnostic and passive immunotherapeutic applications.

Production and purification of proteins used for antibody generation: E.coli expressing recombinant L523S protein were grown overnight in LBBroth with the appropriate antibiotics at 37° C. in a shaking incubator.The next morning, 10 ml of the overnight culture was added to 500 ml of2×YT plus appropriate antibiotics in a 2 L-baffled Erlenmeyer flask.When the optical density (at 560 nanometers) of the culture reached0.4-0.6, the cells were induced with IPTG (1 mM). Four hours afterinduction with IPTG the cells were harvested by centrifugation. Thecells were then washed with phosphate buffered saline and centrifugedagain. The supernatant was discarded and the cells were either frozenfor future use or immediately processed. Twenty milliliters of lysisbuffer was added to the cell pellets and vortexed. To lyse the E. colicells, this mixture was then run through the French Press at a pressureof 16,000 psi. The cells were then centrifuged again and the supernatantand pellet were checked by SDS-PAGE for the partitioning of therecombinant protein. For proteins that localized to the cell pellet, thepellet was resuspended in 10 mM Tris pH 8.0, 1% CHAPS and the inclusionbody pellet was washed and centrifuged again. This procedure wasrepeated twice more.

The washed inclusion body pellet was solubilized with either 8 M urea or6 M guanidine HCl containing 10 mM Tris pH 8.0 plus 10 mM imidazole. Thesolubilized protein was added to 5 ml of nickel-chelate resin (Qiagen)and incubated for 45 min to 1 hour at room temperature with continuousagitation. After incubation, the resin and protein mixture were pouredthrough a disposable column and the flow through was collected. Thecolumn was then washed with 10-20 column volumes of the solubilizationbuffer. The antigen was then eluted from the column using 8M urea, 10 mMTris pH 8.0 and 300 mM imidazole and collected in 3 ml fractions. ASDS-PAGE gel was run to determine which fractions to pool for furtherpurification. As a final purification step, a strong anion exchangeresin such as Hi-Prep Q (Biorad) was equilibrated with the appropriatebuffer and the pooled fractions from above were loaded onto the column.Each antigen was eluted off of the column with an increasing saltgradient. Fractions were collected as the column was run and anotherSDS-PAGE gel was run to determine which fractions from the column topool. The pooled fractions were dialyzed against 10 mM Tris pH 8.0. Thismaterial was then submitted to Quality Control for final release. Therelease criteria were purity as determined by SDS-PAGE or HPLC,concentration as determined by Lowry assay or Amino Acid Analysis,identity as determined by amino terminal protein sequence, and endotoxinlevel was determined by the Limulus (LAL) assay. The protein was thenvialed after filtration through a 0.22-micron filter and the antigenswere frozen until needed for immunization.

To generate anti-L523S mouse monoclonal antibodies, mice were immunizedIP with 50 micrograms of recombinant L523S protein that had been mixedto form an emulsion with an equal volume of Complete Freund's Adjuvant(CFA). Every three weeks animals were injected IP with 50 micrograms ofrecombinant L523S protein that had been mixed with an equal volume ofIFA to form an emulsion. After the fourth injection, spleens wereisolated and standard hybridoma fusion procedures were used to generateanti-L523S mouse monoclonal antibodies.

Anti-L523S monoclonal antibodies were screened by ELISA analysis usingthe bacterially expressed recombinant L523S protein as follows. 96 wellplates were coated with antigen by incubating with 50 microliters(typically 1 microgram) at 4° C. for 20 hours. 250 microliters of BSAblocking buffer was added to the wells and incubated at RT for 2 hours.Plates were washed 6 times with PBS/0.01% tween. Fifty microliters ofeach undiluted monoclonal supernatant were added per well and incubatedat room temperature for 30 minutes. Plates were washed as describedabove before 50 microliters of goat anti-mouse horse radish peroxidase(HRP) at a 1:10000 dilution was added and incubated at RT for 30minutes. Plates were washed as described above and 100 μl of TMBMicrowell Peroxidase Substrate was added to each well. Following a 15minutes incubation in the dark at room temperature the colorimetricreaction was stopped with 100 μl 1N H2504 and read immediately at 450nm. A list of the mouse anti-L523S monoclonal antibodies that weregenerated, as well as their reactivity in an ELISA assay and Westernblot are shown in Table 16. For Western blot analysis, recombinant L523Sprotein was diluted with SDS-PAGE loading buffer containingbeta-mercaptoethanol, then boiled for 10 minutes prior to loading theSDS-PAGE gel. Protein was transferred to nitrocellulose and probed witheach of the anti-L523S hybridoma supernatants. Anti-mouse-HRP was usedto visualize the anti-L523S reactive bands by incubation in ECLsubstrate.

TABLE 16 ELISA and Western Blot Analysis of L523S Monoclonal AntibodiesL523S Mouse Western Blot Monoclonal ELISA HPP14 Supernatant L523SIrrelevant (Irrelevant L523S 213C3 + + ND ND 213C49 + + ND ND 213C62 + +ND ND 213C69 + − − + 213C80 + − − + ND: not determined

The data described in the above example show that L523S is immunogenicand can be used to generate B cell immune responses in vivo. Further,the antibodies generated herein have utility in diagnostic and passiveimmunotherapeutic applications.

Example 35 L523S-SPECIFIC T CELLS AND IDENTIFICATION OF L523S T CELLEPITOPES

This example describes the identification of specific epitopesrecognized by L523S antigen-specific T cells. These experiments furtherconfirm the immunogenicity of the L523S protein and support its use as atarget for vaccine and/or other immunotherapeutic approaches.

A pool of 20-mer peptides, overlapping by 10 amino acids, that span theentire amino acid sequence of L523S (full length amino acid sequence ofL523S provided in SEQ ID NO:176) was used in in vitro culture with Tcells derived from normal donor PBMC to expand CD4 and CD8 T cells.Cultures were established from multiple donors and T cell responses weremonitored following successive in vitro stimulations. L523S-specific Tcell responses were detected in 4 of 4 normal donors. Given that anumber of tumor antigens are identified for each tumor type that arereasonable vaccine candidates, this methodology can be used to comparethe antigen-specific T cell frequency of different antigens.

T cell lines were generated from normal donor PBMCs. The source of Tcells was from the CD69 negative population of PBMCs that had beenprecultured for 1-2 days. Several different priming conditions wereevaluated to identify the most efficient method. These conditions aresummarized in Table 17. In all assays, the T cells were initially primedwith one of the conditions described in Table 17, plus IL-12 for 2-3days. Il-2 and IL-7 were then added to the cultures which were furthercultured for one week. The cultures were then restimulated 2 or 3 timeswith PBMCs pulsed with the entire pool of overlapping L523S peptides.The cells were collected following the last restimulation and analyzedfor antigen-specificity using an IFN-γ solubilized ELISPOT assay. Asshown in Table 17, priming cultures with peptide pulsed dendritic cells(DCs) was the most effective for generating antigen-specific T celllines, either in 96 well or 24 well plates.

TABLE 17 Conditions used to prime donor T cells with L523S overlappingpeptides Condition: A Peptide-pulsed PMBCs/irradiated (overnight pulse,irradiated 11 minutes) B Peptide-pulsed DCs/irradiated (overnight pulse,irradiated 11 minutes) C Peptide-pulsed PBMCs/fixed (overnight pulse, 30second PFA fix) D Peptide-pulsed PBMCs/mitomycin C-treated 30 minutes(overnight pulse) Assay (solubilized Condition (type of plate) ELISPOT)T cell Experiment: Prime Simulation 1 Stimulation 2-3 Target Cellsresponse I A (96U) A (96U) A (24F) D (96U) + II B (96U) A (96U) A (24F)D (96U) +++ III C (96U) C (96U) C (96U) C (96U) − IV B (24F) A (24F) A(24F) D (96U) +++ Abreviations: 96U: 96 well, U-bottomed plates; 24F: 24well, flat-bottomed plates.

Further analysis of the T cell lines generated as described above showedthat these lines generally recognized target cells pulsed with wholeprotein antigen as well as peptides, demonstrating that at least some ofthe epitopes identified are naturally processed. Additional analysisusing anti-MHC Class I and Class II antibodies showed that, while someof the T cell response was MHC class I restricted (CD8+ Tcell-mediated), most of the T cell response generated using this methodwas MHC class II restricted, and thus mediated by CD4+ T cells.

Following the generation of the T cell lines using a pool of overlappingpeptides spanning the entire L523S molecule, target cells pulsed withpools of fewer peptides breaking the L523S into smaller regions werethen used to further map the epitopes recognized by the line generatedfrom 4 different donors. Table 18 summarizes the epitope mappinganalysis using different conditions described in Table 17. The aminoacid sequences of those pools of overlapping peptides that included anepitope (pools 1-6, 14-19, 20-25, 26-30.5, 31-36, 37-40.5, 41-46.5, and47-53) are provided in SEQ ID NO:470-477. Minimal epitope mapping usingT cells from an additional donor, D366, demonstrated that peptide #4(SEQ ID NO:470) was recognized in this donor.

TABLE 18 Summary of L523S epitope analysis Peptide Pool Donor-Condition:1-6 7-13 14-19 20-25 26-30.5 31-36 37-40.5 41-46.5 47-53 D223-IV +D223-I + + + D366-II + + + + D366-I + D446-II + + + D446-I + + D35 +

In an additional study, donor D446 was further evaluated for T cellresponses against 2 other lung-specific antigens in addition to L523S. Tcell lines were generated and epitopes identified from donor D446 usingoverlapping peptides for all three lung-specific antigens. Thisexperiment demonstrated that a single donor can have T cell responses tomultiple antigens, including L523S. In a related study, 3 differentdonors were analyzed for their T cell response to the same lung-specificantigen. All three donors recognized different epitopes of this antigen.Therefore, these data support the use of multiple epitopes from multiplelung tumor antigens, including L523S, in vaccine strategies for lungcancers.

In summary, a peptide pool of overlapping 20-mer peptides spanning theentire L523S protein were used to generate T cell lines and to map Tcell epitopes recognized by these lines. Most, but not all, the T celllines also recognized whole protein pulsed target cells suggesting thatat least some of the epitopes are naturally processed. Furthermore, theresponses to targets pulsed with pooled or individual peptides wereequal or higher than those to target cells pulsed with whole proteinshowing that this technique is more sensitive for detecting immuneresponses. Moreover, this technique can be used for all individuals,regardless of their HLA type. An additional advantage of this approachto evaluating T cell responses to lung-specific antigens is responses toE. coli and viral antigens is avoided. Given that a number of tumorantigens can be identified for a given tumor type that are attractivevaccine candidates, this methodology can be used to compare theantigen-specific T cell frequency of different antigens.

The experiments described above further confirm the immunogenicicty ofthe L523S lung tumor antigen and support its use as a target for vaccineand other immunotherapeutic approaches. Further, the above experimentsidentify specific epitopes of the L523S protein that may be ofparticular importance in the deveolpment of such approaches.

Example 36 VIRAL-MEDIATED DELIVERY OF L523S IN VIVO

This example describes the generation of an illustrative adenovirusvector expressing the L523S lung tumor antigen. This vector was used inin vivo immunogenicity studies, such as those described in Examples 33and 37. This and other viral vectors have utility in DNA-basedvaccination and/or immunotherapeutic strategies for L523S-associatedcancers.

A replication defective E1 and E3 deleted human adenovirus serotype 5vector expressing human L523S under the control of the CMV promoter wasgenerated using standard molecular biology techniques. The cDNA sequenceof the Adenovirus-L523S vector is set forth in SEQ ID NO:479. The cDNAsequence encoding the full-length L523S protein is set forth in SEQ IDNO:478 with the corresponding amino acid sequence set forth in SEQ IDNO:480. Infection of human cells with this construct was shown byimmunoblot analysis to lead to high level expression of the L523Sprotein.

The antigenic nature of the adenoviral proteins introduced into andproduced in the host cell during the course of infection act to increaseimmune surveillance and recognition of L523S as an immunological target.Thus, this adenoviral vector expressing high levels of the L523S tumorantigen has particular utility in inducing therapeutic or curativeimmune responses against endogenous tumor cells expressing L523S in lungcancer patients.

Example 37 IN VIVO IMMUNOGENECITY OF LUNG TUMOR ANTIGEN L523S

This Example further validates the use of L523S DNA and L523S adenovirusprime/boost regimens in generating in vivo immune responses to this lungtumor antigen in vivo. Further, murine CD4 and CD8 T cell epitopes ofhuman L523S are described herein. The results described herein providesupport for the use of L523S DNA and adenovirus prime/boost regimen as avaccine strategy for treating L523S-associated cancers.

The results demonstrated that the vaccination strategy of immunizationwith L523S DNA followed by boosting with L523S adenovirus elicits strongCD4 and CD8 T cell responses as well as antibody responses. Theseresults further showed that C57Bl/6 mice elicit a predominately strongCD8 T cell response, whereas B6D2F1 mice elicit a predominately strongCD4 and antibody response to L523S. The CD8 T cell epitope wasidentified as being contained in the region corresponding to animo acids9-27 (LSENAAPSDLESIFKDAKI; set forth in SEQ ID NO:481) of the L523Sprotein. The epitope was fine mapped to the minimal 9-mer, AAPSDLESI,set forth in SEQ ID NO:491. The CD4 epitope(s) were identified as beingcontained in the region corresponding to animo acids 33-75(FLVKTGYAFVDCPDESWALKAIEALSGKIELHGKPIEVEHSVP; set forth in SEQ IDNO:482) of the L523S protein. Minimal epitopes required for T cellrecognition of both the CD4 and CD8 epitopes are currently beingidentified.

This study involved the immunization of two strains of mice initiallywith L523S naked DNA followed by a second immunization with L523Sadenovirus (see Example 36) two weeks later essentially as described inExample 33. The mice were harvested at day 35 post boost. Table 19describes the immunization regimen of the second study.

TABLE 19 Immunization with L523 DNA Followed by a Second Immunizationwith L523-Adenovirus: Experimental Design Group Prime (d0) Boost (d14)Strain (8/group) 1 100 ug L523S 10⁸ PFU Ad L523 A, B6 DNA*, i.m. i.d 2100 ug L523S 10⁸ PFU Ad L523 A, (B6D2) F1 DNA, i.m. i.d 3 Naïve (B6D2)F1 and B6 PFU = plaque forming unit; Ad = adenovirus. *L523S was clonedinto the pVAX vector (Invitrogen, Carlsbad, CA) using standardtechniques.

Multiple CD8 and CD4 T cell assays were used to determine the in vivoimmunogenicity of L523S DNA/adenovirus prime boost strategy and toidentify the specific epitopes being recognized.

CD8 T Cell Assays:

Pools of overlapping 15-mer peptides spanning the entire L523S proteinwere used to directly stimulate spleen cells. IFNγ producing cells wereanalyzed by IFNγ ELISPOT following a 48 hour stimulation. Thisexperiment showed that strong CD8 T cells specific for L523S peptides 3and 4 within pool 1, corresponding to amino acids 9-27 of the L523Sprotein (LSENAAPSDLESIFKDAKI; set forth in SEQ ID NO:481) were presentin L523S immunized C57bl/6 mice, but not in immunized B6D2F1 mice. Theepitope was fine mapped to the minimal 9-mer, AAPSDLESI, set forth inSEQ ID NO:491. Spleen cells stimulated for 6 hours with pools ofoverlapping peptides were analyzed using intracellular cytokine (IFNγ)staining. Results confirmed that strong CD8 T cell responses weregenerated in C57bl/6 mice to L523S peptide pool 1 but that no responsesto these L523S peptides were observed in immunized B6D2F1 mice.Percentages of peptide specific IFNγ producing CD8 positive T cells inimmunized C57bl/6 mice ranged from 0.17 to 2.86 (media alone controlsranged from 0.02-0.08; naïve controls for peptide pool 1 were at 0.06;irrelevant peptide controls ranged from 0.04-0.07). Chromium releaseassays were then carried out using spleen cells stimulated for 6 dayswith L523S transduced tumor cells as effector cells and peptide-pulsedtumor cells (F45) or tumor cells (EL-4 or RMA) transduced with L523S astargets. Results confirmed that the CD8 T cells specific for L523Speptides present in immunized C57bl/6 mice are functionally lytic.Again, no lytic T cell responses were detected against these L523Speptides in B6D2F1 mice.

CD4 T Cell Assays:

Direct IFNγ ELISPOT assays as described above showed that moderateresponses were detected in B6D2F1 mice. CD4 T cells specific for L523Speptides within pools 3-4, corresponding to amino acids 33-75 of theL523S protein FLVKTGYAFVDCPDESWALKAIEALSGKIELHGKPIEVEHSVP; set forth inSEQ ID NO:482) are present in L523S immunized B6D2F1 mice. No responseswere detected in immunized C57bl/6 mice to these peptides. Intracellularcytokine staining confirmed that IFNγ producing CD4 T cells are presentin L523S immunized B6D2F1 mice. Percentages of peptide specific IFNγproducing CD4 positive T cells in immunized B6D2F1 mice ranged from 0.29to 0.5 (media alone controls ranged from 0.01-0.05; naïve controls forpeptide pool 3/4 were at 0.03; irrelevant peptide controls ranged from0.03-0.08). T cell proliferation and IFNγ production were detected inboth C57bl/6 and B6D2F1 mice following 3 days in vitro stimulation withL523S protein or peptides as shown by standard proliferation and ELISAassays. Peptide pools showing reactivity were the same peptides thatwere detected by IFNγ ELISPOT and intracellular staining assays.

Anti-L523S Antibody Responses:

IgG1 and IgG2a antibody responses to L523S were examined in all groupsof mice essentially as described in Example 33. B6D2F1 mice make stronganti-L523S antibody responses following L523S DNA/adenovirusimmunization, with the IgG2a response being stronger than the IgG1response. Consistent with previous results, C57Bl/6 mice make little tono anti-L523S antibody response following L523S DNA/adenovirusimmunization.

In summary, the results described above further validate the use ofL523S DNA and adenovirus prime/boost regimen as a vaccine strategy. Themouse models described above provide systems for determining theefficacy of DNA/adenovirus L523S immunization strategies and therespective roles of CD4, CD8, and antibody responses in therapeutic andcurative immunity to L523S-expressing tumors. Further, the aboveexperiments further confirm that L523S is immungenic in vivo and thushas utility as a target for vaccine and other immunotherapeuticstrategies.

Example 38 ISOLATION OF A PRIMATE HOMOLOGUE OF L523S

This example describes the isolation of the full-length cDNA and proteinsequence of the rhesus macaque (Macaca mulatta) homologue of the L523Slung tumor antigen. The purpose of this experiment was to identify ananimal model for the validation of L523S vaccine strategies.

Four pairs of PCR primers were designed to anneal to conserved regionsof the L523S cDNA by comparing mouse and human L523S sequences. A rhesusmonkey placenta library was generated using standard techniques and thelibrary cDNA was used as template in a standard PCR reaction. Fouroverlapping amplicons that span the entire L523S cDNA were obtained andsequenced (cDNA set forth in SEQ ID NO:483; amino acid sequence setforth in SEQ ID NO:484). The L523S primate homologue has 99% sequenceidentity to human L523S at the cDNA and amino acid level. Thus, thisexperiment shows that the rhesus macaque provides an animal model inwhich to validate L523S vaccine strategies.

Example 39 EXPRESSION OF FULL-LENGTH L523S IN INSECT CELLS USING ABACULOVIRUS EXPRESSION SYSTEM

This example describes the expression in insect cells of full-lengthlung cancer antigen L523S, together with a C-terminal 10×His Tag, usinga Baculovirus expression system. The recombinant protein has utility inthe development of cancer vaccine, antibody therapeutics and diagnosticsfor cancers associated with L523S expression.

Full-length L523S cDNA, together with its Kozak consensus sequence and aC-terminal 10×His Tag (cDNA set forth in SEQ ID NO:485, amino acidsequence set forth in SEQ ID NO:486), was made by PCR from plasmidPCEP4-L523S, with primers L523F1 (SEQ ID NO:487) and L523RV1, (SEQ IDNO:488). The purified PCR product was cloned into the EcoR I site of thedonor plasmid pFastBac1. The recombinant donor plasmid, pFBL523, wastransformed into E. coli strain DH10Bac (Invitrogen, Carlsbad, Calif.)to make recombinant bacmid in E. coli through site-specifictransposition. The recombinant bacmid DNA was confirmed by PCR analysis,and then transfected into Sf-9 insect cells to make recombinantbaculovirus BVL523. The recombinant virus was amplified to high titerviral stock in Sf-9 cells.

The High Five insect cell line was used to optimize conditions for theprotein expression and for the large-scale production of the recombinantprotein. For the large-scale protein expression, High 5 insect cellswere infected by the recombinant baculovirus at an MOI of 1.0 for 48hours before harvesting. The identity of the protein was confirmed byWestern blot with an affinity-purified rabbit polyclonal antibodyagainst L523S, and by mass spectrometry analysis by CapillaryLC-ESI-MSMS. For Mass-spectrometry analysis, a Capillary column wasfilled with C18 resin (100 mm i.d., 12 cm long). Peptides wereconcentrated on the column and eluted by a gradient of 5 to 65% B over20 min (A: 0.2% acetic acid in water; B: 80% acetronitrile in A). Elutedpeptides were introduced into the ion Trap mass spectrometry (Finnigan,Calif.) by electrospray ionization via an electrospray ionizationinterface (Cytopeia, Seattle, Wash.) and analyzed by data dependent MSand tandem MS (MS/MS) scans. The collision induced dissociation spectra(tandem mass spectra, MS/MS) generated during the experiment weresearched against human protein and L523S protein database using Sequestsoftware to identify possible sequence matches. Using Sequest search, 13peptides from L523S were identified, confirming expression of the L523Sprotein.

Example 40 REGRESSION OF L523S-EXPRESSING MURINE TUMORS FOLLOWINGVACCINATION WITH L523S DNA AND ADENOVIRUS

This Example shows that T cells specific for L523S are capable ofmediating tumor regression in vivo. Therefore, the data described hereinfurther validate the use of L523S DNA and L523S adenovirus prime/boostregimens in generating in vivo immune responses to this lung tumorantigen and provide support for the use of L523S DNA and adenovirusprime/boost regimen as a vaccine strategy for treating L523S-associatedcancers.

The experiments described below demonstrate in vivo efficacy of L523Svaccination in a tumor protection model. The data demonstrate thatvaccination with a combination of VR1012-L523S plasmid DNA (VR1012, see:

Hum Gene Ther 1996 Jun. 20; 7(10):1205-17, Vical Incorporated, 9373Towne Centre Drive, San Diego, Calif.), and recombinant L523S adenovirus(see Example 36) in a prime/boost format can prevent the progression ofL523S-expressing tumor cells in mice. The results show a statisticaldifference in the rate and size of tumors in L523S-vaccinated micecompared to control, naive mice.

C57Bl/6 mice (12/group) were immunized as outlined in Table 20. DNAimmunizations were administered intramuscularly in the anterior tibialismuscle and adenovirus immunizations were administered intradermally atthe base of the tail. On day 28, 7 naïve mice and 8 mice in each of theremaining groups were challenged subcutaneously with 3.0×10⁵ EL4-L523Sstably transduced tumor cells. Tumor growth was monitored every 3-4 daysover the course of the next 3 weeks. The mean tumor size (mm) for eachgroup was measured at each time point. Prior to the final tumormeasurement at 21 days post tumor challenge (day 21), four animals weresacrificed because their tumors were so large. For these animals, themissing tumor measurement at day 21 was estimated using the previous(day 18) tumor measurement. In addition, 4 mice/group were harvested onday 35 for immunologic analysis.

TABLE 20 Immunization with L523S DNA followed by a second immunizationwith L523S-Adenovirus: Experimental Design Primary Immunization BoostGroup Day 0 Day 14 1: L523S DNA/L523S 100 ug pVAX-L523S 10⁸ pfuAdenovirus Adenovirus L523S 2: L523S DNA 100 ug pVAX-L523S — 3: L523SAdenovirus — 10⁸ pfu Adenovirus L523S 4: Naïve — —

Mean tumor size results for Day 15, Day 18, and Day 20 measurements aresummarized in Tables 21, 22, and 23. Table 24 shows the 95% confidencelimits for the difference between mean tumor measurements on day 20(experimental group minus naïve group). The results showed that thedevelopment of tumor in all of the groups including both immunized andnaïve mice appeared to be quite similar until day 10. At that time, thetumor growth in the immunized mice remained constant or regressedslightly whereas the growth of the tumor in naïve mice continued toprogress rapidly. In order to confirm that these observations weresignificant, a statistical analysis was performed as follows. A repeatedmeasures analysis of variance (ANOVA) model, including terms fortreatment group, animal within treatment group and time (day postchallenge) was used to analyze the tumor measurement data. If thetreatment group X time interaction was statistically significant,separate ANOVAs were done for each treatment group and for each time. Ateach time point, each experimental group (Adeno+DNA, Adeno Alone, DNAAlone) was compared to the control group (Naïve) using Dunnett'st-tests. A 0.05 level of significance was used for all analyses.

The statistical analysis showed that there was a significant (p<0.0001)interaction between treatment group and time. Therefore, at each timepoint an ANOVA was performed to compare the treatment groups in terms ofmean tumor size. The treatment groups were not significantly differentat either day 7 (p=0.287) or day 11 post challenge (p=0.570). However,there were significant differences among the treatment groups at theremaining time points (Tables 21-23). The results of this analysisclearly indicate a statistically significant difference in tumor growthbetween the immunized animals and the naïve animals particularly at thelatest time point (day 20).

TABLE 21 Day 15 Tumor Measurements Group N Mean Standard DeviationAdeno + DNA 8 57.4 25.74* Adeno alone 8 49.04 27.99* DNA Alone 8 67.4920.10 Naïve 7 100.24 37.21 *Significantly different from naïve group at0.05 level.

TABLE 22 Day 18 Tumor Measurements Group N Mean Standard DeviationAdeno + DNA 8 59.69 50.58 Adeno alone 8 39.37 36.46* DNA Alone 8 64.1836.95 Naïve 7 121.3 66.9 *Significantly different from naïve group at0.05 level.

TABLE 23 Day 20 Tumor Measurements Group N Mean Standard DeviationAdeno + DNA 8 63.57 56.87* Adeno alone 8 56.48 59.94* DNA Alone 8 66.346.79* Naïve 7 140.25 53.53 *Significantly different from naïve group at0.05 level.

TABLE 24 95% Confidence Limits for Day 20 Tumor Measurements DifferenceBetween 95% Confidence Comparison means Limits DNA Alone - Naïve −73.95(−143.96, −3.93)*  Adeno + DNA - Naïve −76.68 (−146.7, −6.67)* AdenoAlone - Naïve −83.77 (−153.79, −13.76)* *Comparison significant at 0.05level.

In conclusion, the data clearly indicate that T cells specific for L523Sare capable of recognizing and lysing L523S-expressing tumor cell linesin vitro (see Example 37) and that such T cells are capable of mediatingtumor regression in viva Therefore, these data provide support for theuse of L523S DNA and adenovirus prime/boost regimen as a vaccinestrategy for treating L523S-associated cancers.

Example 41 GENERATION OF L514S-SPECIFIC CYTOTOXIC T LYMPHOCYTES (CTL) BYIN VITRO PRIMING AND IDENTIFICATION OF A CTL EPITOPE

This example describes the generation of L514S-specific CD8+ Tlymphocytes from a normal donor and identification of an L514S CTLepitope. L514S is a lung tumor antigen that is preferentially expressedin non small cell lung carcinomas. These experiments further confirm theimmunogenicity of the L514S protein and support its use as a target forvaccine and/or other immunotherapeutic approaches. Further, thisexperiment identifies an illustrative T cell epitope that can be used invaccine and immunotherapeutic strategies.

Autologous dendritic cells were differentiated from Percoll-purifiedmonocytes using GM-CSF (50 ng/ml) and IL-4 (30 ng/ml). Following 5 daysof culture, the dendritic cells were infected with recombinantL514S-adenovirus at an MOI of 20. After infection, the DC were maturedwith the addition of 2 ug/ml CD40L (trimer). CD8+ cells were enrichedfor by the depletion of CD4 and CD14-positive cells. Priming cultureswere initiated in individual wells of six 96-well plates with IL-6 andIL-12. These cultures were restimulated in the presence of IL-2 usingautologous fibroblasts treated with IFN-γ and transduced with L514S andCD80. Following 3 restimulation cycles, the presence of L514S-specificCTL activity was assessed in IFN-γ ELISPOT assays using as APC IFN-γtreated autologous fibroblasts transduced to express either L514S or theirrelevant antigen L552S. Of approximately 576 lines, 8 lines wereidentified that appeared to specifically recognize L514S. Lines 2-4A,3-12E, and 5-3C were cloned using anti-CD3 and feeder cells. The cloneswere tested for specificity on L514S-transduced fibroblasts. In theantibody blocking assay, the fibroblasts transduced with L514S werepre-treated for 30 minutes with the antibody blockers and the finalconcentration was 50 ug/mL once the T cells were added. In theHLA-mismatch assay, the panel of DCs was infected with either adenovirusL514 or a control adenovirus at an MOI of 10. This infection went for 48hours before they were assayed by ELISPOT assay.

To generate CTL, autologous dendritic cells were infected with arecombinant adenovirus that expresses L514S. Purified CD8 T cells werestimulated by these infected DCs and then restimulated weekly usingautologous fibroblasts expressing L514S and the costimulatory moleculeCD80 in the presence of IL-2. Eight microcultures were identified thatspecifically recognize target cells expressing L514S but not controlantigen using ELISPOT analysis. All 8 lines were restimulated andconfirmed by ELISPOT to be specific for L514S. At the same time, threeof the lines were cloned. These lines are referred to as 2-4A, 3-12E,and 5-3C. L514S-specific clones were obtained from all three lines. 50specific clones were obtained from Line 2-4A, 11 specific clones wereobtained from line 3-12E, and 17 specific clones were obtained from line5-3C.

Clones from each line were tested in an antibody blocking assay todetermine their HLA restriction. All of the clones tested appear to beHLA-B/C restricted. Additional experiments using a panel ofadenovirus-L514S and adenovirus control infected DCs that matched atcertain HLA alleles showed that these clones are restricted byHLA*B4403.

In order to map the epitopes being recognized by these clones, clone 6from line 2-4A and clone 1 from line 3-12E were further tested againstautologous PBMC pulsed with 20-mer L514S peptides overlapping by 15amino acids that span the entire L514S protein. Both clones recognizepeptide 28. To fine map the minimal epitope, smaller peptides frompeptide 28 were made and the 10-mer minimal epitope was identified aspeptide 10 (set forth in SEQ ID NO:490; the cDNA encoding this epitopeis set forth in SEQ ID NO:489).

In conclusion, these data confirm the immunogenicity of L514S as a Tcell antigen as well as its suitability as a component of a lung cancervaccine. Further, the above experiments identify a specific epitope ofthe L514S protein that may be of particular importance in thedevelopment of such vaccines.

Example 42 IDENTIFICATION OF ANTIBODIES RECOGNIZING TUMOR ASSOCIATEDANTIGEN NY-ESO-1 PEPTIDE SPECIFIC ANTIGENIC EPITOPES IN BIOLOGICALSAMPLES FROM PATIENTS WITH LUNG CANCER

This example describes the detection of antibodies specific for the lungtumor antigen, NY-ESO-1 in patient serum and lung pleural effusion fluidusing a peptide-array assay. Further, specific epitopes recognized bythese patient antibodies were identified. These data validate the use ofthis peptide-array assay in diagnostic applications.

The peptide-array screening method described in further detail below wasused to characterize a patient's antibodies against one or moreTA-antigens based on antibody specificity, sensitivity (intensity) andclonality. This method was validated by specifically detecting thepresence of antibodies recognizing the tumor-associated antigen(TA-antigen) referred to as NY-ESO-1 (Proc Natl Acad Sci USA 1997 Mar.4; 94(5):1914-8) in lung cancer patient serum and pleural effusion fluidsamples.

In a first study, using Western transfer and immunoblot analysis, serumand lung pleural effusion fluid samples were evaluated for the presenceof antibodies recognizing recombinant NY-ESO-1. Briefly, samples frompatient numbers 205, 208 and 12 were screened by Western transfer andimmunoblot analysis using recombinant NY-ESO-1 protein prepared from anE. coli host cell expression system. The patient serum samples containedantibodies that clearly recognized recombinant NY-ESO-1 6×his-taggedfusion protein (approximately 20 kDa in size), however, the presence ofantibodies recognizing a number of E. coli host cell proteins containedin this preparation of recombinant NY-ESO-1 were also detected.

In order to further characterize a patient's NY-ESO-1 specificantibodies, a series of overlapping peptides 20 amino acids in length,corresponding to the entire sequence of NY-ESO-1, were synthesized anddispensed (displayed) into individual wells of a multiwell plate. Seraor lung pleural effusion samples were then added to each well containingan NY-ESO-1 peptide, negative control wells included buffer alone orpeptides unrelated to TA-antigen NY-ESO-1. An ELISA was used to detectsignal corresponding to the presence of specific antibodies recognizingone or more NY-ESO-1 antigenic epitopes. In this study, the serum sampleobtained from patient sample 205 was shown to contain antibodies capableof detecting an NY-ESO-1 antigenic epitope contained in peptide number2, corresponding to amino acid sequence STGDADGPGGPGIPDGPGGN (SEQ IDNO:492); antibodies contained in patient sample number 208 detectedpeptide number 3, corresponding to amino acid sequencePGIPDGPGGNAGGPGEAGAT (SEQ ID NO:493), peptide number 10, correspondingto amino acid sequence YLAMPFATPMEAELARRSLA (SEQ ID NO:494), peptidenumber 5, corresponding to amino acid sequence GGRGPRGAGAARASGPGGGA (SEQID NO:496) and lower levels of peptide number 2; patient sample number12 recognized peptides 2 and 10; patient sample number 57 recognizedpeptide numbers 2, 5, 10, and 17 corresponding to amino acid sequenceWITQCFLPVFLAQPPSGQRR (SEQ ID NO:495).

Since a number of peptide specific epitopes may be detected by a sampleobtained from a single patient, the peptide-array method disclosedherein is also used to evaluate the clonality of a patient's antibodyrepertoire recognizing a particular TA-antigen. For example, patientsample 205 appears to be monoclonal in its recognition profile, whilepatient sample numbers 12 and 208 appear to be polyclonal in theirrecognition pattern. The clonality of a patient's antibody repertoiremay be used to monitor or otherwise further characterize a patient'sspecific immune response and to evaluate the implications for theprogression of a cancer, such as a lung cancer, in a patient.

In further experiments, Western transfer and immunoblot analysis wasused to confirm that a patient's antibodies recognizing a NY-ESO-1peptide epitope could also detect full-length recombinant NY-ESO-1. Thedata from these experiments clearly indicate that patient sample numbers205 and 12 also detect a full-length recombinant NY-ESO-1 protein. TheNY-ESO-1 signal so detected was shown to be specific, as it was competedaway when immunoblots were probed with patient sample 205 plus peptide2, or when patient sample 12 was used in the presence of peptides 2 and10. Non-specific signal was not competed away in the presence of anyNY-ESO-1 peptide.

Peptides detected according to this procedure were used to search theGenBank protein database for homology with other proteins. Such a searchindicate that NY-ESO-1 peptides 2 and 3 are 100% homologous, peptides 5and 17 are 95% homologous, and peptide 10 is 60% homologous to LAGE-1a,a member of the NY-ESO-1 protein family.

In conclusion, the data described in this example validate thepeptide-array assay by specifically detecting antibodies specific forNY-ESO-1 in lung cancer patients. Further, specific epitopes recognizedby these patient antibodies were described. The disclosed peptide-arrayscreening method has been shown to eliminate detection of non-specificantibodies that may be present in a biological sample derived from apatient, thereby ensuring a high degree of sensitivity and specificity.Peptide-array screening may be used to evaluate the clonality of apatient's antibody repertoire recognizing one or more TA-antigens, andmay be useful in a variety of diagnostic, prognostic and/or therapeuticmethods for lung cancer. Further, the specific epitopes described hereincan also be used in diagnostic, prognostic and/or therapeutic methodsfor lung cancer.

Example 43 IDENTIFICATION OF PATIENT ANTIBODIES RECOGNIZING SPECIFICANTIGENIC PEPTIDE EPITOPES OF THE LUNG TUMOR ASSOCIATED ANTIGEN L523S

This example describes the detection of antibodies specific for the lungtumor antigen, L523S, in lung cancer patient serum and lung pleuraleffusion fluid. Further, specific epitopes recognized by these patientantibodies were identified. Additionally, patient antibodies were shownto crossreact with proteins in the IMP family related to L523S. Thesedata confirm that L523S is immunogenic and support its use to generate Bcell immune responses in vivo. Further, specific peptide epitopes thatcan be used in such approaches were identified. Additionally, thepeptide-array assay described herein can be used in diagnosticapplications for L523S alone or in combination with other lung tumorantigens.

The lung tumor-associated antigen identified in Example 2 as L523S (SEQID NO:175) was shown to be overexpressed in lung cancer tissuesincluding squamous, adenocarcinoma and small cell carcinoma. Recombinant6×his-tagged L523S polypeptide (SEQ ID NO:427) was expressed, purifiedand used in an ELISA to evaluate serum samples obtained from patientswith lung cancer for the presence of antibodies recognizing afull-length L523S polypeptide. The ELISA results indicate that serumsamples from patient numbers 27, 55, 66, and 67 possess antibodiescapable of recognizing recombinant L523S. The same patient serum sampleswere also used in Western transfer (immunoblot) analysis of recombinantL523S 6×his-tagged fusion protein. The serum samples evaluated wereshown to contain specific antibodies capable of detecting recombinantfull-length L523S (approximately 70 kDa) but also contained non-specificantibodies recognizing a number of E. coli proteins that were alsopresent. Similar results were seen using sample number 659-99. Sample 55also contained antibodies that recognized the known lung tumor antigen,NY-ESO-1.

The non-specific detection of, E. coli proteins is unwanted and waseliminated by developing a tumor associated (TA)-antigen specificpeptide-array screening method, which is designed to span the entirelength of the TA-antigen being evaluated, e.g., L523S. To do this, anarray of peptides, representing a series of consecutive overlappingpeptides (20 amino acids in length and overlapping by 10 amino acids)covering the entire length of L5325 were synthesized. Each peptide wasdispersed into individual wells of a multiwell plate and incubated witha serum sample obtained from lung cancer patients. An ELISA was used todetect the presence of antibodies recognizing a specific L523S peptide.The results from this analysis clearly indicated that antibodiescontained in serum samples obtained from numerous lung cancer patientsrecognize L523S peptides as described further below and summarized inTable 25.

Antibodies contained in sera from patient 27 recognize peptide number 42(amino acid sequence KIAPAEAPDAKVRMVIITGP) (SEQ ID NO:497 and SEQ IDNO:548), corresponding to amino acids 440-459 of lung TA-antigen L523S(SEQ ID NO:348). Additionally, competitive Western transfer andimmunoblot analysis was then used to further characterize recognition ofrecombinant L523S by antibodies contained in patient serum samples 27and 659-99. To do this, immunoblots were probed with the serum sampleobtained from patient number 27 in the presence and absence of L523Speptide 42. The results indicate that peptide number 42 blockeddetection of recombinant L523S by antibodies present in samples 27 and659-99. Non-specific detection of E. coli proteins was not competed inthe presence of L523S peptide 42.

Additional sera samples from lung cancer patients as well as normaldonors were also evaluated by peptide-array analysis. In this study,patient sample 13 was also shown to contain antibodies recognizing L523Speptide number 42. Patient sample number 36 recognized peptides 5 (SEQID NO:508), 9 (SEQ ID NO:512) and to a lesser degree peptides 26, 33 and52 (SEQ ID NOs:529, 537, and 559, respectively). The serum sampleobtained from patient 197 contained antibodies strongly recognizingpeptide 17 (SEQ ID NO:520), while peptides 13 (SEQ ID NO:516), 22 (SEQID NO:525) and 52 (SEQ ID NO:559) were detected to a lesser degree.Antibodies contained in lung pleural effusion fluid from patient samplenumber 10 recognized L523S peptide 15 (SEQ ID NO:518), 41 (SEQ IDNO:547), 42 (SEQ ID NO:548), 43 (SEQ ID NO:549) and 53 (SEQ ID NO:560).Lung pleural effusion fluid from patient 14 detected L523S peptidenumber 38 (SEQ ID NO:542). Lung pleural effusion fluid patient samplenumber 15 detected L523S peptides 35 and 53 (SEQ ID NOs:539 and 560,respectively). Lung pleural effusion sample 18 detected L523S peptides42 and 33 (SEQ ID NOs:548 and 537, respectively). A composite multiTA-antigen peptide-array was used to evaluate the humoral response ofpatient number GB-56, detecting L523S peptide 12 (SEQ ID NO:515).Patient samples 208, GB-25, and GB-11 showed no reactivity with L523Speptides indicating that these samples do not contain L523S-specificantibodies. For control samples, serum was obtained from numerous normaldonors (numbers 174, 365, 293, 17, 438, 445, 11, 480). All normal donorswere negative for L523S-specific antibodies except normal donor sample174 that had very low reactivity to peptides 11, 15, 18, 33 and 50 (SEQID NOs:514, 518, 521, 537, and 557, respectively).

In yet another study, a similar peptide array was used to measure thecellular immune response of several samples, essentially as described inExample 35. A cellular response (T cell) of patient number GB-56 wasdetected against a peptide pool containing L523S peptide numbers 30.5-35(SEQ ID NOs:534-539), and another pool that contained peptide numbers36-40.5 (SEQ ID NOs:540-546). A cellular immune response recognizing oneor more peptides contained in the known lung tumor antigen, NY-ESO-1 wasalso detected. In particular, responses against a peptide poolcontaining peptides 13-17 was detected. A cellular immune response ofpatient GB-41 also detected signal in NY-ESO-1 peptide pools containingpeptides 7-12 and 13-17.

TABLE 25 Summary of Antibody Epitopes of L523S Sample/Donor # SampleType L523S peptide Epitope (SEQ ID NOs)  27 Serum 42 (SEQ ID NO: 548),IMP-1 homologue of peptide 42 (SEQ ID NO: 498)  55 Serum IMP-1 homologueof L523S peptide 32 (SEQ ID NO: 502), IMP-2 homologue of L523S peptide32 (SEQ ID NO: 503)  66 Serum Not Determined  67 Serum Not Determined659-99 Serum 42 (SEQ ID NO: 548)  13 Serum 42 (SEQ ID NO: 548), IMP-1homologue of peptide 42 (SEQ ID NO: 498)  36 Serum 5, 9, 26, 33, 52 (SEQID NOs: 508, 512, 529, 537, and 559, respectively) 197 Serum 17, 13, 22,52 (SEQ ID NOs: 520, 516, 525, and 559, respectively)  10 LPE 15, 41,42, 43, 53 (SEQ ID NOs: 518, 547-549, and 560, respectively)  14 LPE 38(SEQ ID NO: 542)  15 LPE 35, 53 (SEQ ID NOs: 539 and 560)  18 LPE 42, 33(SEQ ID NOs: 548 and 537) GB-56 Serum 12 (SEQ ID NO: 515) 208 NoneDetected GB-25 None Detected GB-11 None Detected 287 Serum 32 (SEQ IDNO: 461), IMP-1 homologue of L523S peptide 32 (SEQ ID NO: 502), IMP-2homologue of L523S peptide 32 (SEQ ID NO: 503) 290 Serum 32 (SEQ ID NO:461), IMP-1 homologue of L523S peptide 32 (SEQ ID NO: 502), IMP-2homologue of L523S peptide 32 (SEQ ID NO: 503)

Analysis of the GenBank protein database revealed that the amino acidsequence of L523S (also referred to as IMP-3) peptide numbers 32 and 42share partial homology with the corresponding peptides present in theL523S family members IMP-1 (SEQ ID NO:500) and IMP-2 (SEQ ID NO:501).Patient serum samples 13 and 27, which recognize L523S peptide number 42(SEQ ID NO:548), were also evaluated for their ability to recognize thecorresponding peptides of IMP-1, corresponding to amino acid sequenceKIAPPETPDSKVRMVIITGP (SEQ ID NO: 498) and IMP-2 corresponding to aminoacid sequence KIAPAEGPDVSERMVIITGP (SEQ ID NO:499). The data indicatethat antibodies contained in serum from patient numbers 13 and 27 dorecognize the IMP-1 peptide set forth in SEQ ID NO:498, but not theIMP-2 peptide set forth in SEQ ID NO:499.

In another series of experiments, patient serum sample 55 wascomparatively evaluated for the presence of antibodies recognizing L523Speptide numbers 5 (SEQ ID NO:508), 9 (SEQ ID NO:512), 32 (SEQ ID NO:536)and 42 (SEQ ID NO:548). Cross reactivity with the correspondingpartially homologous peptides from IMP-1 and IMP-2 was also determined.The data indicate that patient sample 55 did not recognize L523Speptides 5, 9 or 42, or the corresponding IMP-1 or IMP-2 peptides; nopeptide in the array was detected by antibodies contained in serumsamples obtained from normal donors, numbers 232 and 481. In addition,patient sample 55 did not recognize L523S peptide number 32, amino acidsequence LYNPERTITVKGNVETCAKA (SEQ ID NO:536)) but did recognize thecorresponding partially homologous IMP-1 peptide corresponding to aminoacid sequence LYNPERTITVKGAIENCCRA (SEQ ID NO:502) and IMP-2 peptidecorresponding to amino acids sequence LYNPERTITVKGTCEACASA (SEQ IDNO:503). Similarly, patient sample numbers 287 and 290 were shown tocontain antibodies that recognize L523S peptide number 32, and whichcross-react at higher titer with the corresponding IMP-1 and IMP-2peptides (the IMP-1 peptide being recognized more strongly than theIMP-2 peptide).

Homology search analysis indicates that L523S (IMP-3) has an overallamino acid sequence identity to IMP-1 and IMP-2 of 74% and 64%,respectively. However, in a similar analysis, L523S peptide number 3(amino acid positions 20-39, SEQ ID NO:506) and peptide 53 (amino acidpositions 560-579, SEQ ID NO:560) were shown to be non-homologous to thecorresponding IMP-1 and IMP-2 peptide regions. Interestingly, patientsera were shown to react with peptide 53 in an additional study.Homology search indicated that peptide 24 of L523S (amino acid positions230-249 (SEQ ID NO:527)) shares significant homology to IMP-1 (80%) andIMP-2 (70%). Lung cancer patient antibodies were also shown to bereactive with peptide 24 in a separate study. Patient sera also reactedagainst peptide 16 (SEQ ID NO:519). In a related study, patient sera wasshown to react with peptides 34 and 37 (SEQ ID NOs:538 and 541).

In conclusion, the data described herein further confirms the in vivoimmunogenicity of the L523S protein and further identifies peptidesrecognized by patient antibodies. These peptides can be used inimmunotherapy or diagnostic applications for cancers associated withover-expression of L523S. The homology between L523S (IMP-3) and thefamily members IMP-1 and IMP-2, along with the cross-reactivity ofantibodies obtained from patient's with lung cancer, suggest that, atleast in some cases, a patient's immune response to L523S may crossreact with IMP family members which are similarly overexpressed in lungtumors relative to normal tissue samples. Additionally, peptide-arrayanalysis as described herein was used to characterize patient antibodyresponses based on antibody specificity, intensity and clonality. Theidentification of antigenic determinants using the peptide-arrayanalysis as set forth herein, is useful for a variety of diagnostic,prognostic and therapeutic methods for lung cancer associated withexpression of L523S either alone or in combination with other lung tumorantigens.

Example 44 GENERATION OF RABBIT POLYCLONAL ANTIBODIES AGAINST L523S ANDIDENTIFICATION OF L523S EPITOPES RECOGNIZED BY THESE ANTIBODIES

This example describes the generation of L523S-specific polyclonalantibodies in rabbits and the identification of specific epitopesrecognized by these antibodies.

Polyclonal antibodies were prepared from rabbits immunized withrecombinant L523S using techniques known in the art and analyzed bypeptide-array for the presence of antibodies recognizing specificTA-antigen L523S antigenic epitopes. Polyclonal antibodies wereL523S-affinity purified from rabbit serum and incubated with an L523Speptide-array as described in Example 43. Specific rabbit antibodiescontained in this polyclonal serum sample strongly recognized peptide 32(SEQ ID NO:536), moderately recognized peptides 16 and 17 (SEQ IDNOs:519 and 520), and to a lesser extent recognized peptides 2, 23, 24,33, 49 and 53 (SEQ ID NOs:505, 526, 527, 537, 556, and 560).

Example 45 IN VIVO GENERATION OF MOUSE POLYCLONAL ANTIBODIES AGAINSTL523S USING A DNA/ADENOVIRUS PRIME BOOST REGIMEN

This example describes the in vivo generation of an L523S-sepcific Bcell response and demonstrates that a DNA/Adenovirus prime-boost regimencan be used to induce a B cell (antibody) response against L523S.Further, specific epitopes recognized by mouse polyclonal antibodies aredescribed.

Polyclonal serum was prepared from mice that were immunized with anL523S DNA/Adenovirus prime boost regimen, essentially as described inExample 33. High titers of mouse antibodies recognizing peptides 17, 22and 53 (SEQ ID NOs: 520, 525, and 560, respectively) were detected. To alesser degree, mouse antibodies recognizing peptides 32, 19 and 18 (SEQID NOs:536, 522, and 521, respectively) were also detected. Antibodiesspecific for adenovirus proteins were also detected that coincided withthe detection of L523S-specific antibodies. No signal was detected usingserum from control naïve mice or mice immunized with an antigenunrelated to L523S. As was shown previously in Examples 33 and 37, bothCD4 and CD8 responses specific for L523S were also detected. Thus, thisexperiment confirms the in vivo immunogenicity of L523S and demonstratesthat a DNA/Adenovirus primer boost regimen can be used to induce a Bcell (antibody) response against L523S.

Example 46 IN VIVO IMMUNOGENICITY OF LUNG TUMOR ANTIGEN L523S: L523S ANDADENOVIRUS-SPECIFIC HUMORAL RESPONSES IN MONKEYS IMMUNIZED WITHL523S-DNA/ADENOVIRUS REGIMEN

This example describes an in vivo immunogenicity study in rhesus macaquemonkeys to evaluate the safety of the vaccine regimen administered astwo priming doses of; VAC/L523S and 2 boosting doses of Ad/L523S.Vaccination with L523S naked DNA was followed by a second immunizationwith either low or high dose of an adenovirus containing L523S. Theresults further validate the use of L523S DNA and L523S adenovirusprime/boost regimens in generating in vivo immune responses to this lungtumor antigen in vivo.

Three groups of monkeys were immunized intradermally. Monkeys wereimmunized three times with DNA followed by three adenovirus-L523Sboosts. Antibody responses to L523S were examined in all groups ofmonkeys on the following days:

−1: one day prior to the 1st DNA-L523S prime

+3: 3 days after the 1st DNA-L523S prime

+31: 2 days after the 3rd DNA-L523S prime

+45: 2 days after the 1st adeno-L523S boost

+73: 2 days after the 3rd adeno-L523S boost

+86: 15 days after the 3rd adeno-L523S boost

The results showed that all pre-bleed monkeys had no antibody titersspecific for L523S and adenovirus particle (SEA-adeno). L523S-DNApriming alone did not induce a significant L523S-specific antibodyresponse. In Group 2 monkeys (monkeys that received low-doseadeno-L523S), 1 of 6 monkeys had a weak antibody response to L523S while6 of 6 monkeys demonstrated a moderate adenovirus-specific antibodyresponse. In Group 3 (monkeys that received high-dose adeno-L523S), 3 of6 monkeys had a strong L523S-specific antibody response while 6 of 6monkeys had a strong adenovirus-specific response. Both male and femalemonkeys generated antibody responses to L523S and adenovirus. Noapparent untoward toxicity was observed in any of the vaccinatedmonkeys.

Thus, the above experiments further confirm that L523S is immunogenic invivo and thus has utility as a target for vaccine and otherimmunotherapeutic strategies. In particular, this study shows that DNAprime followed by high-dose adenovirus boost generates a strong L523Sand adenovirus antibody response.

Example 47 DEVELOPMENT OF AN IN VIVO METASTATIC TUMOR MODEL FOR THEL762P LUNG TUMOR ANTIGEN

This example describes the ability of the lung tumor line 343T/L762P toform three times as many lung tumor foci in CB17 SCID mice as the 343Tparent cell line. This example confirms that L762P (full-length cDNA andprotein sequence set forth in SEQ ID NOs:160 and 161, respectively) is alung tumor antigen and further, shows that the CB17 SCID mouse injectedwith the 343T/L762P cell line is an in vivo model useful for developmentof therapeutics for lung cancers associated with expression of L762P.

The 343T/L762P cell line was generated by transduction of the 343T tumorcell line with a retroviral vector comprising L762P (cDNA set forth inSEQ ID NO:160, amino acid sequence set forth in SEQ ID NO:161), followedby selection resulting in a line that stably expressed L762P, asdescribed further below. Specifically, recombinant retroviruses weregenerated using the Phoenix-Ampho packaging system and the vectors pBiBthat includes a polylinker and the Blasticidin-D selectable marker. ThecDNA for L762P was subcloned into the pBiB vector using standardmolecular techniques. The consensus Kozak sequence GCCACC was includedimmediately 5′ of the initiator ATG to maximize translationalinitiation. As would be recognized by the skilled artisan, any number ofretrovirus vectors available in the art can be used in the context ofthis invention. To characterize surface expression of L762P, theretrovirus construct that expressed L762P was used to transduce the lungtumor cell line 343T. Transduced lines were selected with Blasticidin-Sand expanded to examine L762P surface expression by flow cytometricanalysis. For this analysis, non-transduced and transduced cells werewashed and incubated with 10-50 micrograms/ml of affinity purifiedanti-L762P. Following a 30 minute incubation on ice, cells were washedand incubated with a secondary, FITC-conjugated anti-rabbit IgG antibodyas above. Cells were washed, resuspended in buffer with Propidium Iodide(PI) and examined by flow cytometry using an Excalibur fluorescenceactivated cell sorter. For this analysis, PI-positive (i.e.,dead/permeabilized cells) were excluded. The anti-L762P seraspecifically recognized and bound to the surface of L762P-transducedcells but not the non-transduced counterparts. These resultsdemonstrated that L762P is localized to the cell surface of lung tumorcells.

Two groups of CB17 SCID mice were injected with 4×10⁶ cellsintravenously of either 343T or 343T/L762P cells. Mice were euthanizedafter 42 days and examined for lung tumor foci formation. The averagenumber of foci formation in 343T/L762P-injected mice was 216.8 foci(total for both lungs) as compared to 70.3 in the non-transduced parent343T cell line, giving a ratio of 3.08. Thus, mice injected with theL762P-expressing cell line form three times as many lung tumor foci asmice injected with the parent 343T (non-L762P expressing) cell line.

Example 48 COMPARISONS OF LUNG WEIGHT IN A 343T/L762P METASTATIC TUMORMODEL

This example shows that the ability of the L762P-expressing 343T/L762Pcell line to establish tumors in CB17 SCID mice is significantly greaterthan the non-L762P-expressing parent 343T cell line.

Two groups of CB17 SCID mice were injected with 4×10⁶ cellsintravenously with either 343T/L762P or 343T/EGFP cells. These two celllines were generated by transduction of the 343T tumor cell line with aretroviral vector comprising either L762P (cDNA set forth in SEQ IDNO:160, amino acid sequence set forth in SEQ ID NO:161), or EGFP,followed by selection resulting in cell lines that stably expressedeither L762P or the control marker EGFP, as described in Example 47. Anadditional group of mice received no injections of cells. Mice wereeuthanized after losing 20% of their body weight or 33 days post cellinjection, whichever came first. The average weight of lungs from343T/L762P, 343T/EGFP and uninjected mice were 0.556 g, 0.3 g, and0.1817 g, respectively. Student T-test analysis showed that theprobability of the means of the three groups being equal to one anotherwere as follows: 343T/L762P vs. Uninjected: p=0.0027; 343T/L762P vs.343T/EGFP: p=0.0589; 343T/EGFP vs. Uninjected: p=0.1129.

Thus the 343T/L762P lung tumor cell line, which stably expresses theL762P protein, forms significantly greater mass of lung tumors inintravenously injected CB17 SCID mice as compared to the lungs ofuninjected mice. Additionally, 343T/L762P-injected mice also have lungtumor mass greater than mice injected with the control tumor cell line,343T/EGFP.

Example 49 ADDITIONAL CHARACTERIZATION OF L762P HUMAN MONOCLONALANTIBODIES FACS Binding Analysis

Antibody binding experiments and relative affinity measurements werecarried out to further refine the group of L762P human monoclonalantibodies. Eleven humAbs, selected from those described in Example 31above, were selected for additional characterization. Ranking of therelative binding ability of the humAbs was determined by flow cytometryanalysis, using the ratio of the relative mean fluorescence intensity(MFI) of binding to human L762 transfected 522 cells vs. MFI of bindingto untransfected 522 cells. Briefly, L762P/522 or 522 cells wereharvested and washed in PBS, then incubated with 3ug/ml of the purifiedhumAbs for 30 minutes on ice. After several washes in PBS, 0.5% BSA,0.01% azide, anti-human Ig-PE was added to the cells and incubated for30 minutes on ice. Cells were washed again and resuspended in washbuffer and subjected to flow cytometric analysis. The rankings of thehumAb are shown in Table 26.

Analysis of humAb Binding to the L762P Mouse Orthologue.

HEK 293 cells were transiently transfected with a pCEP4 plasmidconstruct containing the mouse orthologue of the human L762P gene (SEQID NO: 561). Briefly, HEK cells were plated at a density of 100,000cells/ml in DMEM (Gibco, Invitrogen, Carlsbad, Calif.) containing 10%FBS (Hyclone, South Logan, Utha) and grown overnight. The following day,4 μl of Lipofectamine 2000 (Gibco) was added to 100 ul of DMEMcontaining no FBS and incubated for 5 minutes at room temperature (RT).The Lipofectamine/DMEM mixture was then added to 1 μg of L762PFlag/pCEP4 plasmid DNA resuspended in 100 ul DMEM and incubated for 15minutes at RT. The Lipofectamine/DNA mix was then added to the HEK293cells and incubated for 48-72 hours at 37° C. with 7% CO₂. Cells wererinsed with PBS, then collected and pelleted by centrifugation. Cellswere then analyzed by FACS as above. Three of the L762P humAb bound tothe mouse orthologue, see Table 26.

Affinity Analysis

Affinity of the L762P humAbs was determined using Biacore analysis(Biacore, Uppsala, Sweden). Goat anti-human antibody was plated onto aCM5 sensor chip using routine amine coupling. HBS-P running buffer(Biacore) was then used to dilute the L762P humAbs to ˜1 ug/ml, and eachL762P humAb was then captured to a separate channel on the sensor chip.A 5 minute wash was performed to stabilize the L762P humAb baselinereading. L762P antigen (amino acid residues 32-944 of SEQ ID NO:161, ata concentration of 393 nM) was then injected over those surfacescontaining mAbs for 1 minute, followed by a 15 minute dissociation step.To regenerate the binding surface after each capture/inject cycle, a 21second pulse of phosphoric acid (146 mM) was used. The resultingsensograms were then analyzed using the Biacore software and fitted to a1:1 interaction model to determine relative values for Ka and Kd and areshown in Table 26.

Functional Activity

Analysis of apoptosis induction was carried out using L762P/522 orL762P/343 transfected cells (2×10⁵) as described above. The cells wereincubated overnight with 10 μg/ml of the anti-L762P humAbs or irrelevanthuman IgG control mAbs, then assayed for annexin positivity and activecaspase content by incubating the cells with an annexin V-Alexa488conjugate (Molecular Probes, Eugene, Oreg.). Cells were subjected toflow cytometric analysis to determine the amount of annexin positivityas a measure of induced apoptotic activity. Little to no detectableapoptosis above background levels was observed for the humAbs, see Table26.

For anti-proliferation assays, L762P/522 or L762P/343 cells (500cells/well) were plated onto 96 well plates and grown overnight at 37°C., 7% CO₂. The next day, 10-20 μg/ml of the L762P humAbs were added tothe cells and incubated for 6 days at 37 ° C., 7% CO₂. The change in theamount of proliferation was quantitated by the addition of MTS reagent(20 μl/well; Promega, Madison, Wis.) for 1 to 2 hours, followed byreading the OD490 of the plate on a microplate ELISA reader. Little tono reduction in proliferation above background levels was observed forthe L762P humAbs tested (see Table 26).

Internalization Analysis

L762P/522 cells were plated at 1×10³ cells/well in 96 well platescontaining DME plus 10% heat inactivated fetal bovine sera. L762P humAbsor control antibodies, including an irrelevant human IgG and theanti-MHC Class I mAb W6/32, were added at a concentration of 0.5μg/well. A mouse anti-human Ig-saporin conjugated secondary antibody wasthen added at a concentration of 1 μg/ml to the wells, and the plateswere incubated for 6 days at 37° C., 7% CO₂. The decrease in the amountof proliferation was quantitated by the addition of MTS reagent (20μl/well; Promega) for 1 to 2 hours, followed by reading the OD490 of theplate on a microplate ELISA reader. L762P HumAb 1.59.1 caused 15% celldeath and L762P HumAb 2.110.1 caused 20% cell death over the time-courseof this assay, see Table 26.

Antibody Binding Analysis

Saturation binding affinity determination was made using mouse L762PmAb153A12.1 and human L762P humAbs (1.59.1, 2.39.3 and 2.110.1). ThemAbs were radiolabeled with Iodine-125 (Amersham, Arlington Heights,Ill.) using the lodogen method according to manufacturer's instructions(Pierce, Rockford, Ill.). The 153A12.1 mAb was also labeled with In-111(Amersham) after the attachment of a chelator to facilitate Indiumuptake. The labeled mAbs were diluted in binding buffer (PBS/0.5%BSA/1mM sodium azide), then various concentrations of mAbs were added towells of 96-well plates coated with 10 μg/ml of recombinant L762Pprotein (amino acid residues 32-944 of SEQ ID NO:161) or incubated withL762/343T cells.

To determine the total mAb binding, 10 μg/ml L762P antigen or ˜1×10⁶L762P/343T cells were incubated with a dilution series of a particularradiolabeled human or mouse L762P mAb. To determine non-specificbinding, plate-bound L762P antigen or L762P/343T cells were incubatedwith a radiolabeled mAb in the presence of a 50× excess of unlabeledantibody. After 2 to 4 hours incubation at room temperature, plates orcells are washed 4× with cold binding buffer. Washed plates or cellscollected by centrifugation were then subjected to a Gamma counter. Theradioactive signal generated in the non-specific binding sample wassubtracted from the signal derived from the total binding reaction todetermine the specific binding signal. The data were analyzed bynon-linear regression to determine affinity and number of antigen sitesper cell. The derived Kd values are shown in Table 26 and are based onL762P recombinant antigen binding for all of the humAbs tested (1.59.1,2.39.3 and 2.110.1), whereas the mouse L762P mAb (153Al2.1) has anadditional Kd value determination based on binding to L762P/343T cells.

Epitope Mapping using mAbs

Peptide epitopes recognized by mouse monoclonal antibodies 153A12 and153A20 and human monoclonal antibodies humAb 2.4.1 and humAb 2.69.1,were identified using an epitope mapping approach. A series ofoverlapping 20 mer peptides corresponding to the full length amino acidsequence of L762P (SEQ ID NO: 161) were synthesized. As described above,the peptides were subjected to ELISA analysis. The 20 mer L762P peptideswere coated onto flat bottom 96 well microtiter plates at 2 μg/ml andincubated at 37° C. for 2 hours. Plates were then washed 5 times withPBS+0.1% Tween 20 and blocked with PBS+1% BSA for 1 hr. Protein Apurified mouse or human anti-L762P antibodies were then added to thewells at 1 ug/ml and incubated at room temperature for 1 hour. Plateswere again washed, followed by the addition of goatanti-mouse-Ig-horseradish peroxidase (HRP) or goat anti-human-Ig-HRPantibody for 1 hour at room temperature. Plates were washed, thendeveloped by the addition of the chromagenic substrate TMB MicrowellPeroxidase Substrate (Biological Mimetics, Inc., Fredrick, Md.). Thereaction was incubated 15 minutes at room temperature and stopped by theaddition of 1N sulfuric acid. Plates were read at OD450 in an automatedplate reader.

The peptide sequence recognized by L762P mouse monoclonal antibodies153A12 and 153A20 corresponded to aa residues 135-154 of SEQ ID NO:161,EGKYIHFTPNFLLNDNLTAG (SEQ ID NO: 562). The peptide sequence recognizedby L762P human monoclonal antibody humAb 2.4.1 corresponded to aaresidues 571-590 of SEQ ID NO: 161, DKPFYINGQNQIKVTRCSSD (SEQ ID NO:563) and the peptide sequence recognized by humAb 2.69.1 corresponded toaa residues 571-590 of SEQ ID NO: 161, KPGHWTYTLNNTHHSLQALK(SEQ ID NO:382).

Immunohistochemistry Analysis

L762P protein expression was evaluated in various tissues usingimmunohistochemistry (IHC) analysis as described above using mousemonoclonal antibody 153A12.1 (described in Example 31 above).Immunohistochemistry was performed as described herein. Briefly,paraffin-embedded, formalin-fixed tissue was sliced into 8 micronsections. Steam heat induced epitope retrieval (SHIER) in 0.1 M sodiumcitrate buffer (pH 6.0) was used for optimal staining conditions.Sections were incubated with 10% serum/PBS for 5 minutes. Primaryantibody was added to each section for 25 minutes at 1-2 μg/ml, followedby a 25 minute incubation with anti-mouse biotinylated antibody.Endogenous peroxidase activity was blocked by three 1.5 minuteincubations with hydrogen peroxidase. Avidin biotin complex/horse radishperoxidase (ABC/HRP) was used along with DAB chromogen to visualizeantigen expression. Slides were then counterstained with hematoxylin tovisualize cell nuclei. Using this approach, L762P protein was detectedin 11/12 squamous lung cancer, 5/5 squamous esophageal cancer, 5/5squamous skin cancer, 4/5 transition cell carcinoma and 2/5non-neoplastic conditions. No protein was detected in small cell lungcancer, adeno lung cancer, bronchoalveolar cancer, metastatic adeno lungcancer, other cancer of lung and mesothelioma. L762P protein wasdetected in 16/20 stage I lung cancer, 18/20 stage II lung cancer and7/9 stage III/IV lung cancer. L762P protein was detected in 3/4 normallung (bronchia) scattered epithelium, 1/1 normal esophagus epithelium,1/1 normal trachea epithelium, 4/4 normal skin epidermis/sebaceus andsweat glands and 1/1 normal stomach cytoplasmic/gastric pits (lightstaining). No protein was detected in normal thyroid, spleen, lung(alveoli), liver, uterus, prostate, testis, ovary, pancreas, heart,large and small intestine, brain and adrenal tissues.

TABLE 26 FACS Affinity Low Res Functional Saturation Binding BiocoreK_(d) (nM) Activities Binding L762P Human K_(d) Anti- InternalizingK_(d) (nM) K_(d) (nM) humAb (rank) mouse K_(a) (M⁻¹s⁻¹) K_(d) (s⁻¹) (nM)Apoptotic proliferative humAb I¹²⁵ In¹¹¹ 1.41.1 10 − 2.2 × 10⁴ 1.4 ×10⁻⁴ 6.4 — — — — — 1.59.1 2 − 2.3 × 10⁴ 3.8 × 10⁻⁴ 16.0 — — 15% 24.3 —1.98.1 9 + 3.7 × 10⁴ 2.2 × 10⁻⁴ 6.0 — — — — — 1.105.1 7 − 4.6 × 10⁴ 7.8× 10⁻⁴ 17.0 — — — — — 1.259.1 8 − 4.4 × 10⁴ 5.9 × 10⁻⁴ 13.0 — — — — —2.4.1 5 +/− 1.9 × 10⁴ 3.1 × 10⁻⁴ 16.0 — — — — — 2.39.3 6 − 5.0 × 10⁴ 2.3× 10⁻⁴ 4.6 — — — 21.7 — 2.53.3 4 − 3.9 × 10⁴ 2.2 × 10⁻⁴ 5.5 — — — — —2.69.1 11 + 3.5 × 10⁴ 5.0 × 10⁻⁴ 14.0 — — — — — 2.77.1 3 − 5.3 × 10⁴ 2.6× 10⁻⁴ 5.0 — — — — — 2.110.1 1 + 7.9 × 10⁴ 4.1 × 10⁻⁴ 5.3 — — 20%  4.9 —Mouse — − — — — — — — 16.6 18.6 mAb 20.2 153A12.1 (cells)

Example 50 EXPRESSION OF L523S IN ENDOCERVICAL AND ENDOMETRIAL NEOPLASMS

This example demonstrates that expression of the lung tumor antigen,L523S (cDNA set forth in SEQ ID NO:175; amino acid sequence set forth inSEQ ID NO:176), is also significantly increased in adenocarcinoma insitu (AIS) of the uterine cervix and endocervical adenocarcinoma (ECCA)as compared to benign endocervical glands and endometrialadenocarcinoma. Thus, this example shows that L523S can be used as adiagnostic marker for cervical cancer.

Adenocarcinoma in situ (AIS) of the uterine cervix has been recognizedas a precursor lesion for invasive adenocarcinoma. However, there hasbeen very limited molecular evidence to support the concept. L523S hasbeen identified as a carcinoma-associated antigen as shown by itsoverexpression in lung squamous and adeno carcinomas as compared tonormal lung using a variety of techniques including microarray and realtime PCR. The following experiments were done to evaluate the expressionof L523S in AIS, benign endocervical glands (BEG), endocervicaladenocarcinoma (ECCA) and endometrial adenocarcinoma (EMCA).

Cervical biopsies and hysterectomy specimens with AIS (n=20), BEG (n=8),ECCA (n=9) and EMCA (n=25) were obtained. All 20 cases of AIS had BEG inthe same specimen, making a total of 28 samples of BEG available forstudy. Sections were cut at 4 microns and immunostained on an automatedimmunostainer using murine monoclonal L523S-specific antibodies (seeExample 34). Briefly, slides were peroxidase blocked for 5 minutes,steamed for 40 minutes in Citrate Buffer (pH 6.0), cooled for 20minutes, blocked for Avidin/Biotin in Egg/Milk for 15 minutes each,washed in diH₂O, and loaded onto the Autostainer. Positive L523Sstaining was graded as weak (<25% of cells positive) or strong (>25% ofcells positive).

The IHC staining results are summarized in Table 27 and are expressed asthe percentage of cases in each category as shown.

TABLE 27 Immunohistochemical staining of L523S in endocervical andendometrial neoplasms Negative L523S Weak L523S Strong L523S Benign 100(28/28) 0.0 (0/28)  0.0 (0/28)  AIS 10 (2/20) 10 (2/20) 80 (16/20) ECCA11 (1/9)  11 (1/9)  78 (7/9)  EMCA  88 (22/25)  8( 2/25)  4 (1/25)

These results demonstrate significantly increased expression of L523S inAIS and ECCA compared to benign endocervical glands and EMCA, supportingthe concept that AIS is a precursor of invasive carcinoma, and thatL523S may play an important role in AIS and ECCA development andprogression. In addition, L523S expression can be helpful indistinguishing AIS from benign cervical glands, and endocervicaladenocarcinoma from endometrial adenocarcinoma in difficult cases.

In a separate experiment, additional cervical cancer samples wereanalyzed for L523S expression using IHC as described above. The resultsare summarized in Table 28. The results confirm the above study and alsoindicate that L523S expression increases with the stage of cervicalintraepithelial neoplasia (CIN) and may be useful as an early indicatorof cervical cancer development.

TABLE 28 L523S expression in cervical cancers Tissue Diagnosis* StainingResults** Sq. Lung Ca 98-002C v. 1+ blush w/ focal signal Sq. Lung Ca98-002M Negative Sq. Lung Ca 98-002P v. 2+ tumor Sq. Lung Ca 98-012BBuniform 3+ tumor Cervical Ca 98-023F SCC v. 3+ invasive tumor & CIN-3,negative nl Cervical Ca 98-023J SCC Negative Cervical Ca 98-023K SCC v.1-2+ CIN 1-3, negative normal Cervical Ca 98-023L SCC uniform 2-3+invasive tumor Cervical Ca B653/92 SCCUC focal 3+ high grade invasivetumor fragment Cervical Ca B5039/95 SCCUC 1+ CIN3, focal 2+ glandularext. Cervical Ca B173/97 SCCUC V. 2-3+ invasive tumor Cervical CaB1716/97 SCCUC uniform 2-3+ invasive tumor Cervical Ca B3090/97 SCCUCuniform 1-2+ invasive, negative normal fragment Cervical Ca B2176/98SCCUC focal 1+ blush Cervical Ca B2319/98 SCCUC v. 2-3+ invasiveCervical Ca B697/99 SCCUC 1+ blush invasive, negative normal CervixRB98-010G Normal Negative Skin RB01-030A2 Normal no signal abovebackground *SCC: squamous cell carcinoma; SCCUC: squamous cell carcinomauterine cervix **Staining intensities are expressed in increasingintensity as 1+, 2+, or 3+. Blush indicates very light staining. Resultsare further described as focal (less than 25% of cells stain positive),uniform (over 75% of tissue stains positive), or variable (v.: between25% and 75% of tissue stains positive). CIN: cervical intraepithelialneoplasia (precancer). Numbers indicate the stage or grade of theneoplasm, with Grade III being closest to malignancy.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for the treatment of a lung cancer in a patient, wherein thelung cancer is characterized by overexpression of the polynucleotideprovided in SEQ ID NO:347 or a variant of SEQ ID NO:347 having at least95% identity to the polynucleotide sequence of SEQ ID NO:347, comprisingadministering to the patient a polynucleotide comprising thepolynucleotide sequence selected from the group consisting of: (a) thepolynucleotide sequence set forth in SEQ ID NO:347; (b) thepolynucleotide sequence of a variant of SEQ ID NO:347 having at least95% identity to the polynucleotide sequence of SEQ ID NO:347; and (c)the polynucleotide sequence of a fragment of the polynucleotide sequenceof (a) or (b), wherein the fragment encodes an immunogenic portion ofthe polypeptide encoded by the polynucleotide of SEQ ID NO:347.
 2. Amethod for the treatment of a lung cancer in a patient comprisingadministering to the patient the polynucleotide set forth in SEQ IDNO:347, wherein the lung cancer is characterized by overexpression ofthe polypeptide provided in SEQ ID NO:347, or a variant of SEQ ID NO:347having at least 95% identity to the polynucleotide sequence of SEQ IDNO:347.
 3. A method for the treatment of a lung cancer in a patient,wherein the lung cancer is characterized by overexpression of thepolynucleotide provided in SEQ ID NO:347, or a variant of SEQ ID NO:347having at least 95% identity to the polynucleotide sequence of SEQ IDNO:347, comprising administering to the patient a composition comprisinga first component selected from the group consisting of physiologicallyacceptable carriers and immunostimulants and a second componentcomprising a polynucleotide comprising the polynucleotide sequence setforth in SEQ ID NO:347.
 4. The method of claim 1, wherein saidpolynucleotide encodes a fusion protein.
 5. The method of claim 4,wherein the fusion protein comprises a T helper epitope.
 6. The methodof claim 4 wherein the fusion protein comprises a fusion partnerselected from the group consisting of: a protein D derivative, aC-terminal domain of the LYTA protein, Ra12, the non-structural proteinfrom influenza virus (NS1), and an endosomal/lysosomal compartmenttargeting signal.
 7. The method of claim 3, wherein said polynucleotideencodes a fusion protein.
 8. The method of claim 7, wherein the fusionprotein comprises a T helper epitope.
 9. The method of claim 7 whereinthe fusion protein comprises a fusion partner selected from the groupconsisting of: a protein D derivative, a C-terminal domain of the LYTAprotein, Ra12, the non-structural protein from influenza virus (NS1),and an endosomal/lysosomal compartment targeting signal.
 10. The methodof claim 3, wherein said immunostimulant is an adjuvant that induces apredominantly Th1 type immune response.
 11. The method of claim 10,wherein said adjuvant is selected from the group consisting of: 3D-MPL,QS21, a mixture of QS21 and cholesterol, and a CpG oligonucleotide. 12.The method of any one of claims 1-11 wherein the lung cancer is a lungsquamous cell carcinoma or a lung adenocarcinoma.
 13. The method of anyone of claims 1-11 wherein said polynucleotide is delivered by a viralbased delivery system.
 14. The method of claim 13 wherein the viralbased delivery system comprises an adenovirus.
 15. The method of any oneof claims 1-11 wherein said polynucleotide is delivered in a DNAprime/adenovirus boost regimen.